Biofuel and electricity producing fuel cells and systems and methods related to same

ABSTRACT

A fuel cell comprising an anode electrode, a cathode electrode and a reference electrode electronically connected to each other; a first biocatalyst comprising a consolidated bioprocessing organism (e.g., a cellulomonad or  clostridium  or related strains, such as  Cellulomonas uda  (Cuda),  Clostridum lentocellum  (Clen),  Acetivibrio cellulolyticus  ( A. cellulolyticus ),  Clostridium cellobioparum  ( C. cellobioparum ), alcohol-tolerant  C. cellobioparum , alcohol-tolerant Cuda, and combinations thereof) capable of fermenting biomass (e.g., cellulosic biomass or glycerin-containing biomass) to produce a fermentation byproduct; and a second biocatalyst comprising an electricigen (e.g.,  Geobacter sulfurreducens ) capable of transferring substantially all the electrons in the fermentation byproduct (e.g., hydrogen, one or more organic acids, or a combination thereof) to the anode electrode to produce electricity is disclosed. Systems and methods related thereto are also disclosed.

This application is a Divisional of U.S. application Ser. No.13/635,137, filed on Dec. 28, 2012, which application is a U.S. NationalStage Filing under 35 U.S.C. 371 from International Application No.PCT/US2011/028807, filed Mar. 17, 2011, and published in English as WOWO/2011/116185 on Sep. 22, 2011, which claims the benefit under 35U.S.C. 119 (e) of U.S. Provisional Application Ser. No. 61/314,936 filedon Mar. 17, 2010, which applications and publications are herebyincorporated by reference in their entireties.

BACKGROUND

The increased concern for the inevitable depletion of the oil supply aswell as the negative impact of the use of fossil fuels on theenvironment has highlighted the need for biofuel alternatives fromrenewable resources, such as ethanol, diesel, butanol and hydrogen.Ideally, a biofuel should have a high energy content and be compatiblewith the current petroleum-based transportation, storage anddistribution infrastructures.

The inventors recognize a need in the art for systems and methods thatprovide for improved biomass conversion to biofuel in a cost-effectivemanner.

SUMMARY

In one embodiment, a fuel cell comprising an anode electrode, a cathodeelectrode and a reference electrode electronically connected to eachother; a first biocatalyst comprising a consolidated bioprocessingand/or fermentative organism (e.g., a cellulomonad, such as Cellulomonasuda (C. uda), or a clostridium such as Clostridium lentocellum (C.lentocellum), Clostridium cellobioparum (C. cellobioparum), adaptivelyevolved strains of such organisms, such as alcohol-tolerant strains,glycerol-tolerant strains, heat-tolerant strains and combinationsthereof) capable of processing and fermenting biomass (e.g.,cellulosic-containing, polyol-containing, such as glycerin-containingwater, etc.) to produce a biofuel and fermentation byproducts; and asecond biocatalyst comprising an electricity-producing microorganism orelectricigen (e.g., Geobacter sulfurreducens, (Gsu) or alcohol-tolerantGsu (GsuA)) capable of transferring substantially all the electrons inthe fermentation byproducts (e.g., hydrogen, one or more organic acids,or a combination thereof) to the anode electrode to produce electricity.

In one embodiment, the biomass is cellulosic biomass. In one embodiment,the biomass is a polyol, such as glycerin-containing water.

In one embodiment, the fuel cell further comprises an exchange membranecapable of transferring electrons and protons; and an electronic deviceconnected to the anode electrode, the cathode electrode and thereference electrode.

In one embodiment, the anode electrode, the cathode electrode and thereference electrode are located in a single chamber. In one embodiment,the anode electrode and the reference electrode are located in a firstchamber and the cathode electrode is located in a second chamber.

In one embodiment, the fermentation byproduct is primarily hydrogen.

In one embodiment, the consolidated bioprocessing (CBP) organismcomprises one or more cellulomonads, such as Cellulomonas uda (Cuda), orclostridial or a clostridial-related strain, such as C. lentocellum orAcetivibrio cellulolyticus. In one embodiment, the CBP organismcomprises A. Acellulolyticus, C. cellobioparum (Cce) or a combinationthereof. In one embodiment the Cce is a glycerol- or alcohol-tolerantstrain of Cce (CceA) or a combination thereof. In one embodiment,alcohol tolerance is evolved in any of the aforementioned CBP organismsto produce an alcohol-tolerant strain of the CBP organism to improveperformance of the biocatalyst (e.g., alcohol-tolerant Cuda).

Embodiments further include a system comprising a biofuel productionfacility configured to produce a biofuel (e.g., ethanol, biodiesel fuel)and a biomass waste stream (e.g., cellulosic-containing biomass wastestream, glycerin-containing biomass wastestream), wherein the biofuel isproduced from biomass; a fuel cell system configured to produce alcoholand electricity from the biomass waste stream, the fuel cell systemcomprising an anode electrode, a cathode electrode and a referenceelectrode electronically connected to each other; a first biocatalystcomprising a consolidated bioprocessing organism capable of fermentingbiomass to produce a fermentation byproduct; and a second biocatalystcomprising an electricigen capable of transferring substantially all theelectrons in the fermentation byproduct to the anode electrode toproduce electricity. In one embodiment, the system further comprises acomputer system connected to the fuel cell for monitoring andcontrolling fuel cell activity.

In one embodiment, the electrodes are housed in a single chamber or adouble chamber as described above.

Embodiments further include a method comprising, a consolidatedhydrolyzing and fermentation step for converting biomass to a biofuelwith a first organism in an anode chamber, wherein the anode reactorcontains an anode electrode and the converting step produces afermentation byproduct; transferring electrons in the byproduct to theanode electrode with a second organism to produce a film, and allowingthe film to catalytically split the electrons and protons, wherein theelectrons flow towards a cathode electrode to produce electricity andthe protons permeate a proton-exchange membrane connecting the anodechamber and the cathode chamber, wherein the electrons and protons reactto produce hydrogen gas.

In one embodiment, the first and second organisms are addedsequentially. In one embodiment, the first and second organisms areadded substantially simultaneously.

In one embodiment, the method further comprises applying a potential tothe anode electrode.

In one embodiment, the biofuel is ethanol. In one embodiment, theethanol is produced in less than 50 hours at a yield greater than 40% ofa total theoretical yield. In one embodiment, the biofuel is biodieselfuel.

In one embodiment, biomass conversion to biofuel is catalyzed by aconsolidated bioprocessing organism, thus reducing the cost associatedwith enzymatic hydrolysis. In various embodiments, fermentation productsother than the biofuel (i.e., fermentation byproducts, typicallyconsidered to be a waste byproduct) are removed by a second organism,i.e., an electricigen, which converts the fermentation byproducts intoelectricity, thus producing an added-value product. This step alsoprevents media acidification and accumulation of feedback inhibitors andtoxic byproducts, thereby improving hydrolysis and fermentationefficiency.

The biomass may, in some embodiments, be a non-food biomass, such ascorn stover or glycerin-containing water.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a simplified schematic of a consolidated process for ethanoland electricity generation according to an embodiment.

FIG. 2 is a simplified schematic of a microbial fuel cell (MFC)according to an embodiment.

FIG. 3 shows hydrogen (H₂) production versus time for cellobiose andAmmonia Fiber Expansion (AFEX)-corn stover (CS) with Acetivibriocelluloyticus (Ace) or Clostridium lentocellum (Clen) as theconsolidated bioprocessing (CBP) organisms according to variousembodiments.

FIG. 4 is a bar graph showing fermentative growth rates of Clen andCellulomonas uda ATCC 21399 (Cuda) with glucose, xylose, or both,according to various embodiments.

FIG. 5 is a bar graph showing ethanol yields and carbon dioxide (CO₂)yields using AFEX-CS with Geobacter sulfurreducens ATCC 51573 (Gsu) andCuda alone, and in co-culture, in various embodiments.

FIG. 6 shows current versus time for Gsu/acetate and after addition ofAFEX-CS and Cuda according to various embodiments.

FIG. 7 is a bar graph showing fermentation efficiency of sugars,acetate, formate and hydrogen for Cuda alone, and in co-culture withGsu, with and without gas sparging, according to various embodiments.

FIG. 8 is a bar graph showing CO₂ yield and ethanol yield of Gsu and ahydrogen uptake-deficient mutant of Gsu (Gsu Hyb) in co-cultures withCuda according to various embodiments.

FIG. 9 shows current versus time for Gsu/acetate sequentially inoculatedwith Cuda and AFEX-CS at maximum current, zero current, and during adeclining current according to various embodiments.

FIG. 10 shows current versus time for simultaneous inoculations ofAFEX-CS/Cuda/Gsu with acetate (1 mM) and without acetate according tovarious embodiments.

FIG. 11 is a bar graph showing ethanol yield for Cuda, Cuda/Gsu andCuda/Gsu/acetate (1 mM) according to various embodiments.

FIG. 12 is a bar graph showing current yields (as conversion efficiency)for the sequential and simultaneous inoculations of FIGS. 10 and 11,respectively, according to various embodiments.

FIG. 13 is a bar graph showing maximum current yield for the sequentialand simultaneous inoculations of FIGS. 10 and 11, respectively,according to various embodiments.

FIG. 14 is a simplified schematic diagram showing a process forproducing biodiesel fuel.

FIG. 15A is a graph showing glycerol growth of Gsu, C. cellobioparum(Cce), and a co-culture comprising Cce-Gsu, over time according to anembodiment.

FIG. 15B is a bar graph showing fermentation production for Cce and aco-culture comprising Cce-Gsu with ethanol, lactate, acetate, formateand hydrogen according to various embodiments.

FIG. 16A is a graph showing growth rate for Gsu, Cce and Cce-Gsu inglycerol according to various embodiments.

FIG. 16B is a graph showing growth rate for an alcohol-tolerant strainof Gsu (GsuA), a glycerol-tolerant strain of Cce (CceA) and a co-culturecomprising CceA-GsuA in glycerol according to various embodiments.

FIG. 16C is a graph showing growth rate for Gsu, GsuA and CceA inethanol according to various embodiments.

FIG. 17 is a graph showing current versus time for CceA-GsuA grown with10% glycerol according to an embodiment.

DETAILED DESCRIPTION

In the following detailed description of embodiments of the invention,embodiments are described in sufficient detail to enable those skilledin the art to practice them, and it is to be understood that otherembodiments may be utilized and that chemical and procedural changes maybe made without departing from the spirit and scope of the presentsubject matter. The following detailed description is, therefore, not tobe taken in a limiting sense, and the scope of embodiments of thepresent invention is defined only by the appended claims.

In one embodiment, a consolidated bioprocessing technology in abioelectrochemical cell (BEC), such as a microbial fuel cell (MFC), isdriven by first and second microbial partners. The first microbialpartner can be a consolidated bioprocessing (CBP) organism, as definedherein. In one embodiment, the CBP organism degrades a lignocellulosicsubstrate and further co-ferments substantially all the fermentationsugars into ethanol and fermentation byproducts. In one embodiment, thelignocellulosic substrate is pretreated, such as chemically pretreated.In one embodiment, the CBP organism degrades a polyol, such asglycerin-containing water. The second microbial partner can be anelectricigen, as defined herein. In one embodiment, Geobactersulfurreducens (Gsu) serves as the electricigen.

The Detailed Description that follows begins with a definition sectionfollowed by a brief overview of current technologies for production ofalcohol (both grain-based and cellulosic-based), a description of theembodiments, an example section and a brief conclusion.

Definitions

The term “biomass” as used herein, refers in general to organic matterharvested or collected from a renewable biological resource as a sourceof energy. The renewable biological resource can include plantmaterials, animal materials, and/or materials produced biologically. Theterm “biomass” is not considered to include fossil fuels, which are notrenewable.

The term “plant biomass” or “lignocellulosic biomass” as used herein, isintended to refer to virtually any plant-derived organic matter (woodyor non-woody) available for energy on a sustainable basis. Plant biomasscan include, but is not limited to, agricultural crop wastes andresidues such as corn stover, wheat straw, rice straw, sugar canebagasse and the like. Plant biomass further includes, but is not limitedto, woody energy crops, wood wastes and residues such as trees,including fruit trees, such as fruit-bearing trees, (e.g., apple trees,orange trees, and the like), softwood forest thinnings, barky wastes,sawdust, paper and pulp industry waste streams, wood fiber, and thelike. Additionally grass crops, such as various prairie grasses,including prairie cord grass, switchgrass, big bluestem, littlebluestem, side oats grama, and the like, have potential to be producedlarge-scale as additional plant biomass sources. For urban areas,potential plant biomass feedstock includes yard waste (e.g., grassclippings, leaves, tree clippings, brush, etc.) and vegetable processingwaste. Plant biomass is known to be the most prevalent form ofcarbohydrate available in nature and corn stover is currently thelargest source of readily available plant biomass in the United States.

The term “biofuel” as used herein, refers to any renewable solid, liquidor gaseous fuel produced biologically, for example, those derived frombiomass. Most biofuels are originally derived from biological processessuch as the photosynthesis process and can therefore be considered asolar or chemical energy source. Other biofuels, such as naturalpolymers (e.g., chitin or certain sources of microbial cellulose), arenot synthesized during photosynthesis, but can nonetheless be considereda biofuel because they are biodegradable. Biofuels can be derived frombiomass synthesized during photosynthesis. These include, for example,agricultural biofuels (defined below), such as biodiesel fuel. Biofuelscan also be derived from other sources, such as algae, to produce algalbiofuels (e.g., biodiesel fuel). Biofuels can also be derived frommunicipal waste s (residential and light commercial garbage or refuse,with most of the recyclable materials such as glass and metal removed)and from forestry sources (e.g., trees, waste or byproduct streams fromwood products, wood fiber, and pulp and paper industries). Biofuelsproduced from biomass not synthesized during photosynthesis alsoinclude, but are not limited to, those derived from chitin, which is achemically modified form of cellulose known as an N-acetyl glucosaminepolymer. Chitin is a significant component of the waste produced by theaquaculture industry because it comprises the shells of seafood.

The term “biodiesel fuel” or “biodiesel” as used herein, refersgenerally to long-chain (C₁₂-C₂₂) fatty acid alkyl esters, which can beeither fatty acid methyl (FAMEs) or ethyl (FAEEs) esters. Biodiesel fuelcan be produced from both agricultural and algal oil feedstocks.Biodiesel fuel is chemically analogous to petrochemical diesel, whichfuels compression engines and can be mixed with petrodiesel to runconventional diesel engines. Petrodiesel is a fuel mixture of C₉ to C₂₃hydrocarbons of average carbon length of 16, having approximately 75% oflinear, branched, and cyclic alkanes and 25% aromatic hydrocarbons. Ingeneral, biodiesel and petrodiesel fuels have comparable energy content,freezing temperature, vapor pressure, and cetane rating. Biodiesel fuelalso has higher lubricity and reduced emissions. The longer chain inFAEEs increases the cetane rating and energy content of the fuel, whiledecreasing its density, and pour and cloud points. As a result,combustion and flow properties (including cold flow properties) areimproved, as is fuel efficiency. Once combusted, emissions and smokedensities are also minimized.

The term “agricultural biofuel”, as used herein, refers to a biofuelderived from agricultural crops (e.g., grains, such as corn), cropresidues, grain processing facility wastes (e.g., wheat/oat hulls,corn/bean fines, out-of-specification materials, etc.), livestockproduction facility waste (e.g., manure, carcasses, etc.), livestockprocessing facility waste (e.g., undesirable parts, cleansing streams,contaminated materials, etc.), food processing facility waste (e.g.,separated waste streams such as grease, fat, stems, shells, intermediateprocess residue, rinse/cleansing streams, etc.), value-addedagricultural facility byproducts (e.g., distiller's wet grain (DWG) andsyrup from ethanol production facilities, etc.), and the like. Examplesof livestock industries include, but are not limited to, beef, pork,turkey, chicken, egg and dairy facilities. Examples of agriculturalcrops include, but are not limited to, any type of non-woody plant(e.g., cotton), grains such as corn, wheat, soybeans, sorghum, barley,oats, rye, and the like, herbs (e.g., peanuts), short rotationherbaceous crops such as switchgrass, alfalfa, and so forth.

The term “glycerin-containing water”, as used herein, refers to aliquid, such as water, containing any amount of glycerin (i.e.,glycerol, a polyol). The liquid can contain other components, such assolids, alcohols, oils, salts and/or other components.Glycerin-containing water includes glycerin wastewater produced as awaste product of biodiesel fuel production or from ethanolbiorefineries. Although glycerin wastewater can refer to either “crudeglycerin wastewater” (i.e., “unrefined glycerin wastewater” which isglycerin wastewater in its initial state after separation from abiodiesel fuel product) or “refined glycerin wastewater” followingtreatment (typically in preparation for selling) which increases thevolume of glycerin to at least about 80% by volume, the processesdescribed herein are useful with crude glycerin water, thus eliminatingthe need for refining glycerin wastewater in the conventional manner.

The term “biodegradable”, as used herein, refers to a substrate capableof being decomposed, i.e., chemically broken down, by the action of oneor more biological agents, such as bacteria.

The term “electricigen” as used herein, refers to a biocatalyst which iselectrochemically active or an electricity-generating microorganism,i.e., an organism capable of transferring electrons to an electrode withor without mediators.

The term “bioprocessing microorganism” as used herein, refers to amicroorganism capable of degrading biomass, such as lignocellulosicbiomass.

The term “consolidated bioprocessing (CBP) organism” as used hereinrefers to a biocatalyst which is also capable of fermenting the degradedbiomass into one or more biofuels, i.e., capable of performing a singlestep hydrolysis and fermentation.

The term “alcohol-tolerant” as used herein, refers to a mutant of amicrobial strain adaptively evolved or genetically engineered to haveincreased tolerance to alcohol as compared with the native microbe.

The term “glycerol-tolerant” as used herein, refers to a mutant of amicrobial strain adaptively evolved or genetically engineered to have anincreased tolerance to glycerol as compared with the native microbe.

The term “heat-tolerant” as used herein, refers to a mutant of amicrobial strain adaptively evolved or genetically engineered to have anincreased tolerance to heat as compared with the native microbe.

The term “adaptive evolution” as used herein, refers to the process thatenhances the fitness of an organism to a particular environmentalcondition under appropriate selective pressure.

The term “ethanologenesis”, as used herein, refers to the metabolicprocess that results in the production of ethanol.

The terms “fuel cell” or “electrochemical cell”, as used herein, referto a device used for the generation of electricity from a chemical ormicrobial reaction. The reaction can proceed naturally or can befacilitated with electrical input from, for example, a potentiostat. Afuel cell is comprised of anode and cathode electrodes connected througha conductive material. The electrodes may be housed in a single ordouble, i.e., separate chamber and when housed in a double, i.e.,separate chamber, may be separated by a cation- or proton-exchangemembrane. A chemical or biological catalyst added to the anode driveselectricity generation.

The term “microbial fuel cell” or “microbial electrochemical cell” asused herein, refers to a fuel cell driven by electricigenicmicroorganisms either in a substantially pure (i.e., at least 90%purity) culture of at least a single species or in a mixed-speciesculture, i.e., a co-culture, which can include the electricigen at anyconcentration and a number of other species or as part of microbialconsortia, i.e., a group of different species of microorganisms whichmay have different metabolic capabilities, but which act together as acommunity, such as a natural (e.g., biofilms) or defined laboratorymicrobial consortia.

The term “bioelectrochemical cell” or “BEC” as used herein, refers to anMFC capable of inputting additional voltage to control product outputsof the system and increase its performance.

The term “bioelectricity” as used herein, refers to electricity producedbiologically, e.g., from biological materials such as biofuels andbiomass.

The term “pretreatment step” as used herein, refers to any step intendedto alter native biomass so it can be more efficiently and economicallyconverted to reactive intermediate chemical compounds such as sugars,organic acids, etc., which can then be further processed to a variety ofvalue added products such a value-added chemical, such as ethanol.Pretreatment can reduce the degree of crystallinity of a polymericsubstrate, reduce the interference of lignin with biomass conversion andprehydrolyze some of the structural carbohydrates, thus increasing theirenzymatic digestibility and accelerating the degradation of biomass touseful products. Pretreatment methods can utilize acids of varyingconcentrations (including sulfuric acids, hydrochloric acids, organicacids, etc.) and/or other components such as ammonia, ammonium, lime,and the like. Pretreatment methods can additionally or alternativelyutilize hydrothermal treatments including water, heat, steam orpressurized steam. Pretreatment can occur or be deployed in varioustypes of containers, reactors, pipes, flow through cells and the like.Most pretreatment methods will cause the partial or full solubilizationand/or destabilization of lignin and/or hydrolysis of hemicellulose topentose sugars.

The term “moisture content” as used herein, refers to percent moistureof biomass. The moisture content is calculated as grams of water pergram of wet biomass (biomass dry matter plus water) times 100%.

The term “Ammonia Fiber Explosion” or “Ammonia Fiber Expansion”(hereinafter “AFEX”) pretreatment” as used herein, refers to a processfor pretreating biomass with ammonia to solubilize lignin and redepositit from in between plant cell walls to the surface of the biomass. AnAFEX pretreatment disrupts the lignocellulosic matrix, thus modifyingthe structure of lignin, partially hydrolyzing hemicellulose, andincreasing the accessibility of cellulose and the remaininghemicellulose to subsequent enzymatic degradation. Lignin is the primaryimpediment to enzymatic hydrolysis of native biomass, and removal ortransformation of lignin is a suspected mechanism of several of theleading pretreatment technologies, including AFEX. However in contrastto many other pretreatments, the lower temperatures and non-acidicconditions of the AFEX process prevents lignin from being converted intofurfural, hydroxymethyl furfural, and organic acids that couldnegatively affect microbial activity. The process further expands andswells cellulose fibers and further breaks up amorphous hemicellulose inlignocellulosic biomass. These structural changes open up the plant cellwall structure enabling more efficient and complete conversion oflignocellulosic biomass to value-added products while preserving thenutrient value and composition of the material. See, for example, themethods described in U.S. Pat. Nos. 6,106,888, 7,187,176, 5,037,663, and4,600,590, all of which are hereby incorporated by reference in theirentirety as if fully set forth herein.

Grain-Based Alcohol

The methods for producing various types of alcohol from grain generallyfollow similar procedures, depending on whether the process is operatedwet or dry. One alcohol of great interest today is ethanol. Ethanol canbe produced from virtually any type of grain, but is most often madefrom corn. Corn contains high levels of starches that can be broken downinto the glucose sugars needed for traditional fermentation.

The corn grain may be chemically pretreated, as is known in the art, toincrease its enzymatic digestibility. Thereafter, the pretreated corngrain is hydrolyzed into soluble sugars by microbial enzyme mixtures.The resulting soluble sugars are only partially fermented by geneticallyengineered strains of bacteria or fungi. Fermentation is also limited byacidification of the media, due, in part, to the production of organicacids during microbial fermentative metabolism. Fermentation is alsolimited by feedback inhibition on the microbial metabolism fromfermentation byproducts such as hydrogen gas and organic acids.

The resulting ethanol is then distilled and evaporated for separationfrom the remaining fermentation products. The solid biomass wastematerial is filtered and washed to separate the lignin waste from theremaining biomass waste. Optimization efforts have focused ongenetically engineering microbes for degrading and fermenting thebiomass substrate, cellulase improvement by direct evolution, and/orengineering industrial strains with more efficient and stable cellulaseand hemicellulase enzymes, with only moderate success. However,production of alcohols (e.g., ethanol) using grain is gradually beingreplaced with other, more efficient and sustainable solutions. Of note,production of ethanol as described above, also generates a glycerolbyproduct at concentrations up to about 10% (w/w) of the total sugarconsumed.

Biomass Conversion to Alcohol

Nearly all forms of lignocellulosic biomass, i.e., plant biomass, suchas monocots, comprise three primary chemical fractions: hemicellulose,cellulose, and lignin. Hemicellulose is a polymer of short,highly-branched chains of mostly five-carbon pentose sugars (xylose andarabinose), and to a lesser extent six-carbon hexose sugars (galactose,glucose and mannose). Dicots, on the other hand, have a high content ofpectate and/or pectin, which is a polymer of alpha-linked glucuronicacid. Pectate may be “decorated” with mannose or rhamnose sugars, also).These sugars are highly substituted with acetic acid.

Because of its branched structure, hemicellulose is amorphous andrelatively easy to hydrolyze (breakdown or cleave) to its individualconstituent sugars by enzyme or dilute acid treatment. Cellulose is alinear polymer of glucose sugars, much like starch, which is the primarysubstrate of corn grain in dry grain and wet mill ethanol plants.However, unlike starch, the glucose sugars of cellulose are strungtogether by β-glycosidic linkages which allow cellulose to formclosely-associated linear chains. Because of the high degree of hydrogenbonding that can occur between cellulose chains, cellulose forms a rigidcrystalline structure that is highly stable and much more resistant tohydrolysis by chemical or enzymatic attack than starch or hemicellulosepolymers. Lignin, which is a polymer of phenolic molecules, providesstructural integrity to plants, and remains as residual material afterthe sugars in plant biomass have been fermented to ethanol. Lignin is aby-product of alcohol production and is considered a premium qualitysolid fuel because of its zero sulfur content and heating value, whichis near that of sub-bituminous coal.

Typical ranges of hemicellulose, cellulose, and lignin concentrations inplants are shown inhttp://www1.eere.energy.gov/biomass/feedstock_databases.html. Typically,cellulose makes up 30 to 50% of residues from agricultural, municipal,and forestry sources. Cellulose is more difficult to hydrolyze thanhemicellulose, but, once hydrolyzed, converts more efficiently intoethanol with glucose fermentation than hemicellulose. In contrast, thesugar polymers of hemicellulose are relatively easy to hydrolyze, but donot convert as efficiently as cellulose using standard fermentationstrains (which produce ethanol from glucose). Although hemicellulosesugars represent the “low-hanging” fruit for conversion to ethanol, thesubstantially higher content of cellulose represents the greaterpotential for maximizing alcohol yields, such as ethanol, on a per tonbasis of plant biomass.

Conventional methods used to convert biomass to alcohol includeprocesses employing a concentrated acid hydrolysis pretreatment, atwo-stage acid hydrolysis pretreatment as well as processes employingany known conventional pretreatment, such as hydrothermal or chemicalpretreatments, followed by an enzymatic hydrolysis (i.e.,enzyme-catalyzed hydrolysis) or simultaneous enzymatic hydrolysis andsaccharification. Such pretreatment methods can include, but are notlimited to, dilute acid hydrolysis, high pressure hot water-basedmethods, i.e., hydrothermal treatments such as steam explosion andaqueous hot water extraction, reactor systems (e.g., batch, continuousflow, counter-flow, flow-through, and the like), AFEX, ammonia recycledpercolation (ARP), lime treatment and a pH-based treatment. However,pretreatment-hydrolysis of plant biomass can often result in thecreation and release of other chemicals that inhibit microbialfermentation. These inhibitors (i.e. furfural) are largely the productof sugar degradation, and methods to remove these inhibitors or toreduce their formation or strains resistant to the inhibitors areneeded.

Several of these methods generate nearly complete hydrolysis of thehemicellulose fraction to efficiently recover high yields of the solublepentose sugars. However, chemical solubilization of hemicellulose alsoproduces toxic products, such as furan derivatives, which can inhibitdownstream microbial reactions (e.g., fermentation). Regardless, thehydrolysis of hemicellulose facilitates the physical removal of thesurrounding hemicellulose and lignin, thus exposing the cellulose tolater processing. However, most, if not all, pretreatment approaches donot significantly hydrolyze the cellulose fraction of biomass.

Biomass conversion to alcohol also poses unique fermentationconsiderations. The Saccharomyces cerevisiae yeast strains used inconventional corn ethanol plants for example, can ferment glucose, butcannot ferment pentose sugars such as xylose. Additionally, there iscurrently no naturally occurring microorganism that can effectivelyconvert all the major sugars present in plant biomass to ethanol.Therefore, genetically engineered yeast or bacteria, which can, intheory, ferment both glucose and xylose to alcohol, are being used forbiomass to alcohol processes. However, in practice, co-fermentation inthis manner is inefficient and glucose fermentation is still the mainreaction for ethanol production. Furthermore, genetically-enhancedrecombinant strains of fermentative microorganisms, includingrecombinant strains of yeast, bacteria and fungi, as well as transgenicnucleic acids (DNA, RNA) derived from such components may poseenvironmental disposal and permitting problems.

Biodiesel Fuel Production

Biodiesel fuel (hereinafter “biodiesel”) is typically produced viatransesterification of triglycerides using an alcohol and catalyst. Inthe presence of the catalyst, the alcohol reacts with the oil'striglycerides and sequentially removes one methyl ester at a time togenerate biodiesel fatty acid esters and glycerol as shown in thereaction below:

Biodiesel's fatty acid esters have variable lengths and bondscorresponding to the side chains of the triglycerides in the startingoil, with the most frequent being palmitate (C16:0), stearic acid(C18:0), oleic acid (C18:1), linoleic acid (C18:2), and linolenic acid(C18:3) in different proportions. Although acid and alkali catalysts canbe used for the transesterification reaction, many commercial biodieselproducers use alkaline catalysts, which are less corrosive and have ahigher (about 4,000 times) reaction rate. Inexpensive alkaline catalystssuch as sodium and potassium hydroxide are commonly used atconcentrations between about 0.5% and about 1% to achieve yields ofbiodiesel ranging from about 94 to about 99%.

Methanol and ethanol are the most common alcohols used in thetransesterification reaction and produce, respectively, fatty acidmethyl esters (FAMEs) and fatty acid ethyl esters (FAEEs). As comparedto methanol, ethanol is more biodegradable and has a lower toxicity.Ethanol further has a higher solubility as compared to methanol,allowing for higher reaction temperatures and increases in the reactionrate.

As shown in FIG. 14, biodiesel production involves not onlytransesterification, but also separation of the crude biodiesel fromglycerin waste, and biodiesel refining. In an example biodieselproduction process, transesterification can proceed in a reactor forabout 1-2.5 hrs at about 60 to about 80° C. to generate an approximately10:90 mixture of crude glycerin (i.e., a mixture of glycerol, alcohol,catalyst and oil impurities) and crude biodiesel. The higher density ofcrude glycerin enables a phase separation in a settling tank or acentrifuge. Water can be added to the mix to improve the phaseseparation, thus generating a crude glycerin stream of roughly about40-50% glycerol, together with some alcohol, oils, most of the catalyst,and soap.

Crude biodiesel also contains impurities (primarily soap and catalyst,together with alcohol), and is typically further refined. The alcohol,for example, can be removed with water, thus producing high-qualitybiodiesel. Thereafter, the washed wet biodiesel is allowed to dry, suchas by a vacuum flash process, while the wastewater, containing alcohol,salts and glycerol, is removed by suitable means, such as withcentrifugation, and added to the crude glycerin waste. This step furtherdilutes the concentration of glycerol in the glycerin wastewater to ca.10-20% with an average alcohol content of 5%.

Glycerin wastewater is the major waste product of the biodiesel industryand can pose environmental concerns, as it is generally not costeffective to refine and concentrate the glycerol in the wastewater tosell to glycerol refineries. As FIG. 14 shows, prior to selling glycerinwastewater, costly pretreatment and concentration steps are generallyundertaken. As such, unrefined glycerin wastewater is oftentimesdisposed of as hazardous waste (e.g., containing hazardousconcentrations of glycerol and methanol), which can be costly to thebiodiesel producer.

As shown in FIG. 14, glycerin wastewater is generally refined prior toselling, to remove oils, alcohol and salts before being concentrated toa purity of approximately 80% glycerol, a concentration generallyrecognized in the industry as a minimum standard for glycerol feedstockpurity. This is a costly process that involves acid pretreatment toremove oils and to neutralize and precipitate the alkali-catalyst andconvert the soaps into water-soluble salts and free fatty acids. Evenmore costly is the stripping of the low concentrations of alcohol fromthe glycerin solution and its concentration by flash-evaporation ordistillation.

DESCRIPTION OF THE EMBODIMENTS

A novel system for producing alcohol and generating electricity in acombined or consolidated process is described herein. In one embodiment,the process involves providing biomass, such aslignocellulosic-containing biomass (such as from an ethanol productionfacility) or a polyol-containing biomass, such as glycerin-containingwater, such as glycerin wastewater (e.g., crude or partially refinedglycerin wastewater from a biodiesel production facility).

In the embodiment shown in FIG. 1, a process 100 is provided in whichlignocellulosic-containing biomass 102 is subjected to one or morepretreatment steps to separate lignin 106 from insolublecellulose/hemicellulose (hereinafter “insolubles”) 108.

In the embodiment shown in FIG. 1, the insolubles 108 are provided to amicrobial fuel cell (MFC) 118 where they are degraded, i.e., hydrolyzedand fermented in a single-step process using a consolidatedbioprocessing (CBP) microbe 110 to produce ethanol 112 and fermentationbyproducts such as hydrogen gas (H₂) 114 and organic acids 116. Thehydrogen gas 114 and/or organic acids 116 provide a source of electrons124 to support the growth of an electricigen 120, which gains energy bytransferring electrons 124 to an electrode 126, thereby producingelectricity 124 and a carbon dioxide (CO₂)-containing waste stream 122.

Unlike conventional cellulosic ethanol processes which require separatehydrolysis and fermentation steps, embodiments described herein providefor use of a CBP organism 110 which is not only capable of catalyzingthe enzymatic hydrolysis, but can also serve as an alcohologenicbiocatalyst (alcohologenesis). In one embodiment, the CBP organism 110serves as an alcohologenic biocatalyst (e.g., an ethanologenicbiocatalyst). As such, the embodiments described herein are not relianton a previous biomass solubilization step or previous growth of the CBPorganism 110 and electricigen 120 in separate vessels prior toinitiation of the fermentation process. This is in contrast withconventional methods in which the electricigenic organism is grown as apure culture on an electrode of a first fuel cell to produce anelectrochemically-active film, which is then transferred to a secondfuel cell inoculated with an ethanologenic microbe and supplemented witha biomass hydrolysate. Such steps not only add complexity to theprocess, they increase costs. Furthermore, the use of ethanologenicmicroorganisms in conventional methods that produce fermentationbyproducts other than those that the electricigen can convert intoelectricity results in reduced electricity production and feedbackinhibition of the fermentation by the CBP organism 110. In embodimentsdescribed herein, both the CBP organism 110 and the electricigen 120 canbe simultaneously inoculated or sequentially inoculated in the samereactor while maintaining the net production of ethanol and electricity(See Example 3).

Any suitable biomass, as defined herein can be used. In one embodiment,the biomass is a non-food biomass, such as agricultural waste. In oneembodiment, corn stover is used. Additional steps known in the art mayalso be used to prepare the biomass for use in the novel process,including, but not limited to, milling.

The pretreatment step 104 may take the form of any known pretreatmentstep, including chemical pretreatment. Heating or cooking with addedwater may also occur, as is known in the art. In a particular embodimentAFEX corn stover as defined herein, is used. Any fuel cell 118 having asuitable configuration and size may be used in the embodiments describedherein. In one embodiment, the fuel cell is a microbial fuel cell (MFC),such as the type described in Microbial Fuel Cells—Challenges andApplications, Bruce E. Logan and John M. Regan, Environ. Sci. Technol.,2006, 40 (17), pp 5172-5180, which is incorporated herein by referencein its entirety.

In one embodiment, the anode and cathode electrodes are housed inseparate chambers. In one embodiment, the anode and cathode electrodesare separated by a cation- or proton-exchange membrane. Spacing betweenthe anode electrode and the cathode electrode can also vary, as isunderstood by those skilled in the art.

In one embodiment, the anode and cathode electrodes of the fuel cell arehoused in the same chamber. See, for example, Microbial biofilmvoltammetry: direct electrochemical characterization of catalyticelectrode-attached biofilms. Marsili E, Rollefson J B, Baron D B,Hozalski R M, Bond D R. Appl Environ Microbiol. 2008 December:74(23):7329-37. Epub 2008 Oct. 10, which is hereby incorporated byreference in its entirety.

In one embodiment, an external or air-breathing cathode electrode isused. In this embodiment, the cathode chamber is removed and the cathodeelectrode is placed externally and in direct contact with theproton-exchange membrane. See, for example, Improved fuel cell andelectrode designs for producing electricity from microbial degradation,Park D H, Zeikus J G. Biotechnol Bioeng. 2003 Feb. 5:81(3):348-55 andElectrically enhanced ethanol fermentation by Clostridium thermocellumand Saccharomyces cerevisiae. Shin H S, Zeikus J G, Jain M K., ApplMicrobiol Biotechnol. 2002 March: 58(4):476-81, both of which areincorporated herein by reference in their entireties.

In one embodiment, the electrode materials are selected from any knownconductive material, including, but not limited to, carbon, precious ornon-precious metals, metal-organic compounds, stainless steel,conductive polymers, and the like, further including combinationsthereof. In one embodiment, the cathode electrode material and the anodeelectrode material are different materials. In one embodiment, eachelectrode can have any suitable configuration as is known to thoseskilled in the art, with each electrode having the same or a differentconfiguration, as desired. In one embodiment, each electrode has aconfiguration selected from one or more sheets (made from any conductivematerial), or one or more of various types of cloth, paper, glass, brushand rods, and the like, or any combination thereof. Further details ofone embodiment of a MFC are shown in FIG. 2.

In one embodiment, the CBP organism 110 not only hydrolyzeslignocellulosic substrates and produces ethanol at high yields (greaterthan 40% of maximum theoretical yield), but further primarily producesfermentation byproducts (including, for example, primarily organic acidsand/or primarily hydrogen gas and/or primarily other fermentationbyproducts known in the art), which are used as electron donors forgrowth of and electricity generation by an electricigen. In oneembodiment, the CBP organism 110 is a microbe in the clostridial orcellulomonad groups. In one embodiment, Cellulomonas uda ATCC 21399(hereinafter “Cuda” or “C. uda”) is used, which produces primarilyacetate and formate in addition to ethanol. The acetate and formate areconverted into electricity by the electricigen Geobacter sulfurreducens(hereinafter “Gsu” or “G. sulfurreducens”). This process removes organicacids from the growth medium, which prevents media acidification andfeedback inhibition of C. uda's fermentative metabolism. As a result, C.uda's growth and ethanol production are stimulated in the co-culture.Furthermore, because substantially all fermentation byproducts areconverted into electricity, substantially all electrons potentiallyavailable for electricity generation are recovered and ethanol is theonly fermentation product remaining in the liquid medium.

This is in contrast to certain known microbes, such as Clostridiumcellulolyticum, which do not hydrolyze or ferment sufficiently, and alsoproduce a wide range of fermentation byproducts, including those thatcannot be converted into electricity by the electricigen. Such aplatform leads to electron losses and accumulation of ‘waste’fermentation byproducts, rather than net production of ethanol andelectricity, as desired. As such, the embodiments described herein donot include use of Clostridium cellulolyticum as the CBP organism 110.

Any suitable electricigen 120 may be added to the anode chamber 202 todrive electricity generation. In one embodiment, the electricigen 120produces conductive protein filaments termed “pilus nanowires” thatallow substantial stacking of cells on the electrode and efficientelectron flow across the electricigenic film and to the electrode. Thisincludes, but is not limited to, members of the Geobacteraceae family,such as G. sulfurreducens.

In one embodiment, a cathodic chemical reaction, such as an oxygen orferric cyanide oxidation reaction occurs in the cathode chamber. Such anembodiment may be used in applications where electronic input from apotentiostat is not feasible or cost-efficient.

Ethanol yields are expected to be higher than 30% of the totalfermentation product. In one embodiment, the yield may be higher than40%, 50%, 60%, 70%, 80%, 90% or higher, including any range therebetween.

This is in contrast to previous attempts by others to produce bothethanol and electricity (such as with C. cellulolyticum and partiallyamorphous cellulose, e.g., cellulose/hemicellulose, rather thaninsolubles 108), in which ethanol yields less than 40% are obtained, dueto relatively inefficient conversion of fermentation byproducts intoelectricity. Such methods further require the electricigen to bepreviously grown in a separate microbial fuel cell. However, in analternative embodiment, the electricigen (e.g., 120) and/or the CBPorganism (e.g., 110), may optionally be grown in a separate microbialfuel cell although again, this is not required.

The novel systems and methods described herein are efficient, completingthe one-step hydrolysis and fermentation process to produce a maximumethanol yield with a desired CBP organism (e.g., 110) in a time periodof less than about 50 hours. Generally, the time period is less thanconventional methods of reaching a maximum ethanol yield throughseparate hydrolysis and fermentation steps, which can take more than 100hrs, such as up to 120 hrs.

In one embodiment, ethanol yields of at least 80% of the maximumtheoretical yield is produced after less than 50 hours, such as about 40to 46 hours, approximately 43 hours and 80% of the maximum yields areproduced after less than 50 hours, such as at least about 45 hours.

In the embodiment shown in FIG. 2, a MFC 118 is provided, which is anelectrochemical cell comprising two chambers (i.e., anode chamber 204and cathode chamber 205) in an “H” configuration. An anode electrode 206is located in the anode chamber 204 and a cathode electrode 207 islocated in the cathode chamber 205. A cation- or proton-exchangemembrane 210, together with gaskets 211 and glass flanges 212, create a“glass bridge” which separates the anode and cathode chambers, 204 and205, respectively.

In this embodiment, each chamber, 204 and 205 contains an amount ofgrowth medium 208, which, in one embodiment, is substantially identical.The growth medium 208 can be any medium that supports growth of thebiocatalysts 224, and do not necessarily need to be the same in eachchamber, 204 and 205. In one embodiment, the growth medium 208 is freshwater (FW) (See Lovley, D. R., and E. J. P. Phillips. 1988. Novel modeof microbial energy metabolism: organic carbon oxidation coupled todissimilatory reduction of iron or manganese. Appl Environ Microbiol.54(6): 1472-1480), which is incorporated herein by reference in itsentirety.

In one embodiment, the growth medium 208 further contains minerals,vitamins, or combinations thereof. In one embodiment, “Regan's medium”is used as the growth medium 208. (See Ren, Z., T. E. Ward, and J. M.Regan. 2007. Electricity production from cellulose in a microbial fuelcell using a defined binary culture. Environ. Sci. Technol. 41:4781-6,hereinafter “Regan”) which is incorporated herein by reference in itsentirety. In one embodiment, “Daniel Bond's medium” is used as thegrowth medium 208. (See Marsili, E., Rollefson, J. B., et al., 2008,Microbial biofilm voltammetry: direct electrochemical characterizationof catalytic electrode-attached biofilms. Appl Environ Microbiol.,December:74(23):7329-3), which is hereby incorporated herein byreference in its entirety. In one embodiment, the growth medium 208 ispresent in the anode chamber 204 and cathode chamber 205 in substantialquantities so all the electrodes are fully immersed.

The anode electrode 206 and the cathode electrode 207 are electronicallyconnected via anode conductive wires and cathode conductive wires, 213Aand 213B, respectively, both of which, in turn, are connected to apotentiostat 214. The anode chamber 204 further houses a referenceelectrode 216, which is also connected to the potentiostat 214 withconductive wires 213C, as shown in FIG. 2.

The anode chamber 204 and cathode chamber 205 are sealed with an anodestopper 218A and a cathode stopper 218B, respectively. An anode outletport 220A is provided in the anode stopper 218A and a cathode outletport 220B is provided in the cathode stopper 220B. The anode chamber 204is further equipped with an anode sparging port 222A into which a firstneedle 223A can be inserted. Similarly, the cathode chamber 205 isequipped with a cathode sparging port 222B into which a second needle223B can be inserted. The sparging ports, 222A and 222B, further includesuitably sized stoppers, as is known in the art.

In use, the potentiostat 214 poises the anode electrode 206 at a definedpotential, thus allowing for a cathode-unlimited system for controlledand reproducible results. In one embodiment, the process begins byadding a quantity of biomass insolubles 108 (e.g., pretreated cornstover) and a quantity of each biocatalyst 224 (i.e., one or moreelectricigens 120 and one or more CBP organisms 110, as shown in FIG. 1)to the anode chamber 204 to initiate biomass processing.

The insolubles 108 can have any suitable moisture content. In oneembodiment, the moisture content is at least about 15%. The insolubles108 may be dried prior to use, if, for example, they have been storedfor a period of time, although such a step increases the cost of theprocess. Likewise, both biocatalysts 224 can take any form, including asolid or liquid. In one embodiment, at least one of the biocatalysts 224is added as a substantially concentrated wet cell pellet. In oneembodiment, it is added as a dry (lyophilized) powder.

The biocatalysts 224 can be added at substantially the same time orsequentially, as noted herein. As the single step hydrolysis andfermentation of the biomass insolubles 108 proceeds, ethanol 230 isproduced in the anode chamber 204. In one embodiment, the ethanol 230 isgas-stripped from the growth medium 208 of the anode chamber 204 via theanode outlet port 220A and collected in another vessel or pipe as it isbeing produced, for immediate or for later distribution. In theembodiment shown in FIG. 2, ethanol 230 produced as a result of thefermentation is discharged through the anode outlet port 220A, althoughthe invention is not so limited. Ethanol 230 can be drawn off in anysuitable manner, including in a liquid phase.

In one embodiment, the anode outlet port 220A also allows carbon dioxide(CO₂) to be vented out of the MFC 118 during the fermentation portion ofthe single step hydrolysis and fermentation. In one embodiment, the CO₂is collected and recycled for use in an off-site process.

Fermentation byproducts comprising primarily one or more organic acids(not shown) and an amount of hydrogen (Hf) produced with the singlehydrolysis and fermentation step are exposed to a second biocatalyst 224(i.e., electricigen 120, as shown in FIG. 1) causing an electricigenicfilm 228 to grow on the anode electrode 206. The electricigenic film 228can grow to any suitable thickness. In one embodiment, theelectricigenic film 228 is at least about 40 to about 50 micrometersthick.

The electricigenic film 228 catalyzes the split of electrons (e⁻) andprotons (H⁺) present in the fermentation byproducts, causing theelectrons (e⁻) to flow from the anode electrode 206 towards the cathodeelectrode 207 (such as through conductive wires 213A, into thepotentiostat 214, and into conductive wires 213B, as shown in FIG. 2).The protons (H⁺) permeate the proton-exchange membrane 210 and reactwith the electrons (e⁻) at the cathode electrode 207, thereby generatinghydrogen gas (H₂). In the embodiment shown in FIG. 2, the hydrogen gas(H₂) generated in the cathode chamber 205 exits through the outlet port220B.

Both sparging ports, 222A and 222B, are configured to remove oxygen gas,facilitate mixing, and/or provide defined gases for buffering the pH ofthe growth medium (e.g., CO₂-containing gas to buffer the pH ofbicarbonate-containing medium) from their respective chambers, 204 and205, and, ultimately from the MFC 118. Mixing also can be achieved withstir bars 224, as is known in the art.

In one embodiment, glycerol-containing water is used as feedstock togenerate ethanol and/or electricity in a microbial electrochemical cell.The glycerin-containing water can be subjected to one or morepretreatment steps to remove unwanted components, such as oils and salts(while retaining both glycerol and, if present, alcohol). In oneembodiment, the pretreatment additionally or alternatively includes aconcentration step to increase concentration of the glycerol in theglycerin-containing wastewater to a desired level.

In this embodiment, therefore, the “insoluble” component (comparable toinsolubles 108 in FIG. 1) is glycerin. As such the glycerin can beprovided to a BEC, such as a MFC, (e.g., the MFC 118 shown in FIG. 1).The glycerin is then degraded, i.e., hydrolyzed and fermented in asingle-step process using a consolidated bioprocessing (CBP) microbe(e.g., 110) to produce an alcohol (e.g., ethanol 112) and fermentationbyproducts such as hydrogen gas (H₂) and organic acids. The hydrogen gas114 and/or organic acids provide a source of electrons to support thegrowth of an electricigen as described in FIGS. 1 and 2. Again, asdescribed herein with lignocellulosic biomass, glycerin-containingbiomass embodiments described herein provide for use of a CBP organismwhich is not only capable of catalyzing the enzymatic hydrolysis, butcan also serve as an alcohologenic biocatalyst (alcohologenesis). In oneembodiment, the CBP organism 110 serves as an ethanologenic biocatalyst.(See further discussion above with respect to FIGS. 1 and 2, which isapplicable to the glycerin embodiment).

Additionally, glycerol is a permeant solute which enters freely inside acell, thus affecting the properties of the intracellular aqueousenvironment and enzymatic processes, which can lead to growthinhibition. Furthermore, the viscosity of the medium also increases athigh glycerol loading and microbial cells can be osmotically stressed.In one embodiment glycerol tolerance of microbial catalysts useful asalcohologenic biocatalysts is increased by at least two fold up to about10 fold. In one embodiment, the improvement is between about four andsix fold. In one embodiment, the glycerol tolerance of microbialcatalysts useful as alcohologenic biocatalysts is increased at leastabout 10-fold. (See also Example 5). With further modification of themicrobial cells, it is possible the improvement may be even higher than10-fold.

In one embodiment, the alcohologenic biocatalyst is Clostridiumcellobioparum (Cce) which can ferment glycerol into ethanol andfermentation byproducts which can be converted into electricity with Gsuas the electricigen. In one embodiment, a glycerol-tolerant strain ofCce (CceA) or an alcohol-tolerant strain of Gsu (GsuA) or a co-cultureof Cce-Gsu, CceA-GsuA or any combination thereof, including anycombination with Cce, is used as the alcohologenic biocatalyst (SeeExample 5).

In one embodiment, allyl alcohol selection is used to further improvethe performance of an alcohol-tolerant catalyst. In one embodiment,selective pressure on a glycerol tolerant catalyst, such as CceA, can beincreased to accelerate the selection process by selecting for mutantsin the ethanol dehydrogenase enzyme. This enzyme catalyzes the naturalconversion of acetaldehyde to ethanol in clostridia but also convertsallyl alcohol into acrolein. The presence of allyl alcohol in the growthmedium is expected to rapidly select for variants that produce mutantethanol dehydrogenase isoenzymes with diminished affinity for allylalcohol while maintaining or increasing their affinity for acetaldehyde.As a result, these variants are expected to have high ethanol toleranceand high ethanologenic rates

In one embodiment, this approach can be used to accelerate the selectionfor ethanol-tolerant strains of CceA with improved fermentative ratesand higher ethanol yields. Variants have also been reported to arisethat carry mutations that reduce the activity of the acetaldehydedehydrogenase enzyme, which catalyzes the conversion of acetyl-CoA toacetaldehyde. However, these strains can be differentiated because theyhave not evolved alcohol tolerance and have reduced ethanol yields. Suchvariants can be prevented from growing by supplementing the growthmedium with not only allyl alcohol, but ethanol. Thus, it is expectedthat the chances of isolating the desired variants can be increased byadding ethanol to the cultures as well.

Inasmuch as Gsu does not have the ethanol dehydrogenase enzyme, adifferent approach using elevated temperatures can be followed toincrease the selective pressure for alcohol-tolerant strains of GsuA. Inone embodiment, the incubation temperature of the chosen strain can begradually increased from any suitable starting point lower than theoptimal temperature for growth up to just above the optimal temperaturefor growth. In one embodiment, the incubation temperature can begradually increased, starting at about 37° C. up to about 40° C. (e.g.,2° C. above the optimal temperature for growth of Gsu (an exampleelectricigen) and Cce (an example CBP), respectively).

In one embodiment, growth can be monitored as optical density andcultures can also be transferred in stationary phase to capitalize onthe error-prone behavior of DNA Polymerase IV. Growth rates are expectedto decrease at first as suboptimal temperatures are used. However,maintaining the temperature selection is expected to eventually selectfor variants that have recovered the original growth rates. At thispoint, aliquots of the cultures can be preserved at a suitabletemperature, such as down to about −80° C. The cultures can then betransferred and incubated at a higher temperature at the desired time.Eventually, it is expected that temperatures in which optimal growthrates do not recover can be reached, thus marking the end of theadaptive evolution. The heat-tolerant strains can then be tested foralcohol tolerance, which is expected to have increased at the ancestralgrowth temperature.

As described in the Examples below, the inventors are the first toprovide a method for adaptively evolving glycerol tolerance inalcohologenic biocatalysts. In one embodiment using Cce as thebiocatalyst, successive passages at increasing concentrations ofglycerol can allow various strains to grow at high glycerol loads of upto about 10%, further up to about 20%. In one embodiment, CceA is grownat glycerol loads of up to about 10%.

In one embodiment, the ethanol tolerance of CceA is improved whilesimultaneously improving the glycerol tolerance, as the ethanolsensitivity of CceA can mask its true ability to grow and ferment higherloads of glycerol. In one embodiment, cells are grown with increasingconcentrations of glycerol until their optical density stabilizes, whichis a signal that the cells have entered a stationary phase of growth. Atthis phase, mutation rates are highest and the pressure to use glycerolselects for mutant variants with highest growth rates. In oneembodiment, variants show improved growth, allowing them to outcompetethe slower cells, and can be enriched in successive transfers. In oneembodiment, positive selection of these variant proceeds, thus allowingthe cultures to reach stationary phase faster.

Once the growth rates stabilize, in one embodiment, the cultures canagain be transferred and allowed to grow to an early exponentiallyphase, where the most rapidly growing cells will predominate. In oneembodiment, clonal variants can be isolated on glycerol-containingplates and grown in fresh liquid medium to an exponential phase. Aftersuccessive transfers in exponential phase the fastest growers can beselected and preserved at a suitable temperature, such as a temperaturedown to about −80° C. In one embodiment, the variant with the highestgrowth rates and yields can be used to initiate a new round of adaptiveevolution at the next glycerol increment.

In one embodiment alcohologenic biocatalysts in a co-culture areadaptively evolved to co-evolve traits of interest. In one embodiment,adaptively evolving a co-culture (i.e., more than one culture, such asCceA and GsuA), exerts a multiple selection on each component in theco-culture to tolerate higher glycerol loads, to ferment glycerolfaster, to increase alcohol tolerance (as more alcohol accumulates), andto remove and utilize the fermentation byproducts (including lactate).Thus, a co-culture can be evolved to grow at increasing glycerolloadings. In one embodiment, a rapid fluorescence assay is used, whichallows performance monitoring of a co-culture as a function of catalystgrowth. In one embodiment, cell numbers of each of the strains in aco-culture are quantified by the assay by initially staining all cellsin one color (e.g., green) with a suitable binding dye, such as anucleic acid-binding dye SYTO 9 and counter-staining Gram-negative cellsin another color (e.g., red) with a suitable acid-binding dye, such asfluorescent nucleic acid-binding dye hexidium iodide. See, for example,Haugland, R. P. Molecular Probes. Handbook of fluorescent probes andresearch chemicals., (1999), which is incorporated herein by referencein its entirety.

The differential absorption and emission of the two dyes enables theirrapid detection and quantification in a fluoroplate reader. In oneembodiment, dye intensity for the cell numbers of each strain in theco-culture is standardized and can measure the absolute cell number foreach strain and the ratio of the two. The ratios are expected to beconstant if no variants arise or if variants of the two arisesimultaneously. Conversely, the ratios will likely change when variantsof one or the other arise first. Growth can be routinely monitored asoptical density. Those co-cultures showing improved growth rates can beanalyzed with the fluorescence assay described above to quantify thecatalysts' growth and calculate the ratios.

In one embodiment, the co-cultures follow a step-wise evolution sincethe chances of one positive variant arising in only one member arehigher than the probability of positive and complementary mutationsarising in the two microbial catalysts simultaneously. For example, CceAvariants with increased fermentation rates may arise first, which canproduce more fermentation products. This can add selective pressure onGsuA to tolerate higher concentrations of growth inhibitors (e.g.,ethanol or lactate) and remove electron donors faster. Thus, GsuAvariants with enhanced tolerance, uptake and/or metabolism offermentation products can be selected for in successive transfers. Thiscan result in increased cell numbers for GsuA and recovery of the ratiosof the two microbial catalysts. In one embodiment, when variants arise,aliquots are plated in solid medium supplemented with a suitable amountof glucose, acetate and/or fumarate to isolate individual quantities,i.e., an amount sufficient to support enough doubling times from asingle cell so the colony is visible to the naked eye. In oneembodiment, about 0.2% (w/v) glucose is used for CceA and about 0.2%(w/v) acetate and fumarate is used for GsuA. Thereafter, clonalselection, growth, storage, and tests for alcohol and glycerol toleranceand fermentation can be performed as described in the example section.

In one embodiment, genetic engineering is used to improve performance ofthe alcohologenic biocatalyst. Although adaptive evolution can be usedto improve the electrical conversion of lactate by Gsu, the availabilityof a genetic system for this organism enables the application of geneticengineering tools as well. The genetic basis of Gsu's inefficientlactate utilization has been well studied. Strains of Gsu adaptivelyevolved to grow in lactate media with doubling times comparable to thepreferred electron donor, acetate, have been isolated and their genome,sequenced. Summers, Z. M. et al. in Genomics: GTL Contractor-GranteeWorkshop VII, (ed. Office of Science U.S. Department of Energy) 121(Genome Management Information System (Oak Ridge National Laboratory)),which is incorporated herein by reference in its entirety. All of thelactate-adapted strains had single base-pair substitutions in a gene(GSU0514) encoding a repressor of the succinyl-CoA synthetase enzyme.This enzyme catalyzes the conversion of succinyl-CoA to succinate in theTCA cycle when acetate is not the electron donor. Because the GSU0514repressor also regulates the activity of other genes, it is important toselect for single point mutations that activate the succinyl-CoAsynthetase gene without disrupting the normal functioning of the cell.In one embodiment, random mutagenesis can be performed by rolling circleerror prone PCR, a method in which the PCR-amplified GSU0514 is firstcloned into the GsuA expression vector pRG5 and then amplified in itsentirety under error-prone conditions to introduce random mutations.See, for example, Fujii, R., Kitaoka, M. & Hayashi, K. One-step randommutagenesis by error-prone rolling circle amplification. Nucleic AcidsRes. 32, e145, doi:32/19/e145 [pii] 10.1093/nar/gnh147 (2004) and Coppi,M. V., Leang, C., Sandler, S. J. & Lovley, D. R. Development of agenetic system for Geobacter sulfurreducens. Appl. Environ. Microbiol.67, 3180-3187 (2001) (hereinafter “Coppi”), both of which areincorporated by reference in their entireties.

In one embodiment, the plasmid mix can then be electroporated in aGSU0514-deletion mutant of GsuA using recombinant PCR techniques andmutants of interest can be isolated based on their ability to grow onsolid medium with lactate as sole electron donor. (See Coppi). In oneembodiment, the mutants with the fastest growth rates can be introducedinto GsuA via recombinant PCR to generate stable mutants, which can thenbe tested in a BEC powered with lactate as an electron donor (SeeCoppi).

In one embodiment, the “H” configuration MFC (shown in FIG. 2) with theanode electrode poised to a fixed potential is used. In one embodiment,lactate removal from the medium can be monitored by HPLC using a UVdetector. The electrical conversion of lactate can then be calculated ascoulombic efficiency (the amount of usable electrons in the lactateconsumed (12 electrons per mol) divided by the electrons measured ascurrent). In one embodiment, coulombic efficiencies similar to thepreferred electron donor, acetate, (such as about 80% up to about 90%),are reached.

In one embodiment, a mutant of CceA defective in lactate production isgenetically engineered. The mutation can be introduced into a selectedadaptively evolved strain, such as one showing high alcohol tolerance,glycerol loading, and growth robustness. Clostridia are known to producelactate in a single reaction from pyruvate that is catalyzed by thelactate dehydrogenase enzyme. See, for example, Yazdani, S S., et al,Anaerobic fermentation of glycerol: a path to economic viability for thebiofuels industry, Current Opinion in Biotechnology, Volume 18, Issue 3,June 2007, Pages 213-219, which is incorporated herein by reference inits entirety. Although the genome sequence of Cce is not available, manyother closely-related, clostridial genomes are. See, for example,Collins, M. D. et al. The phylogeny of the genus Clostridium: proposalof five new genera and eleven new species combinations. Int. J. Syst.Bacteriol. 44, 812-826 (1994), which is incorporated herein by referencein its entirety.

In one embodiment, the high conservation of clostridial lactatedehydrogenase genes can allow alignment of the sequences andidentification of regions of conservation for the design of degeneratePCR primers. These primers can be used to amplify the Cce lactatedehydrogenase gene, which will be sequenced. A universal genetic systemis available for targeted mutagenesis in clostridia, which allows forthe generation of stable insertion mutants in just a few (10 to 14)days. See, for example, Heap, J. T., Pennington, O. J., Cartman, S. T.,Carter, G. P. & Minton, N. P. The ClosTron: a universal gene knock-outsystem for the genus Clostridium. J. Microbiol. Methods 70, 452-464,doi:S0167-7012(07)00208-4 [pii] 10.1016/j.mimet.2007.05.021 (2007)(hereinafter “Heap 2007”) and Heap, J. T. et al. The ClosTron:Mutagenesis in Clostridium refined and streamlined. J. Microbiol.Methods 80, 49-55, doi:50167-7012(09)00350-9 [pii]10.1016/j.mimet.2009.10.018 (2010) (hereinafter “Heap 2010”), both ofwhich are herein incorporated by reference in their entireties. Themethod, known as the ClosTron, consists of a plasmid with a bacterialgroup II intron sequence where a short sequence of the targeted gene iscloned. The plasmid is introduced in the bacterium by electroporationand positive clones are selected in the presence of the plasmid'santibiotic. See, for example, Phillips-Jones, M. K. in Electroporationprotocols for microorganisms Vol. 47 (ed J. A. Nickoloff) Ch. 23,227-235 (Humana Press Inc., 1995), which is incorporated herein byreference in its entirety. In one embodiment, the CceA minimuminhibitory concentration to the antibiotics available in the variousClosTron plasmids is established (See Heap 2007 and Heap 2010). In oneembodiment, the specific ClosTron target is synthesized for the lactatedehydrogenase. The plasmid replicates in the clostridial host andconstitutively expresses the intron, which spontaneously inserts itselfinto the chromosome at the targeted location. The clones with an introninsertion become resistant to a second antibiotic and can be easilyisolated on selective plates. The plasmid is later lost producing astable insertion in the gene of choice. In one embodiment, the lactatemutant of CceA can be grown with glycerol to confirm it does not producelactate. Because more pyruvate is available for acetate and ethanolproduction, higher current (from acetate) and/or ethanol yields areexpected.

Ethanol production in clostridia proceeds in two reactions catalyzed bythe acetaldehyde and ethanol dehydrogenase enzymes. In one embodiment,an ethanol-deficient mutant of Cce may be produced. In one embodiment,genetic engineering is used to inactivate the first reaction toeffectively reroute the acetyl-CoA towards the synthesis of acetate. Theacetyl-CoA to acetate reaction generates ATP and is expected to beenergetically favored. The result is an ethanol-deficient mutant thatpredominantly ferments glycerol to acetate. In one embodiment, themutation can be introduced in a lactate-deficient CceA strain togenerate a mutant that ferments glycerol to acetate, formate and H₂only. Because these are the electron donors that have the highestelectrical conversion rates by Gsu, current production in a MFC isexpected to increase.

Electrochemical parameters can be further improved to increase theefficiency of the platform in the MFC. In one embodiment, the voltagepotential of the MFC, for example, can be adjusted to promote efficientelectrical conversion of the fermentation byproduct mix.

In one embodiment, the power density obtainable with the glycerolembodiment is improved as compared to conventional microbial fuel cells.In one embodiment, the efficiency of glycerol removal is also improvedas compared to conventional microbial fuel cells. In one embodiment, thecolumbic efficiency of the glycerol embodiment is also improved ascompared to conventional microbial cells. In one embodiment, theconcentration of glycerol used is greater than 2.5% by volume, such asup to about 10% or higher, up to about 20%, about 30%, about 40%, about50%, about 60%, about 70%, about 80%, about 90%, up to substantiallypure glycerol, including any ranges there between. In one embodiment,the concentration of glycerol in the glycerin-containing water is atleast about 50% up to about 80%. In one embodiment, the electrogenicactivity is improved as compared to a conventional microbial fuel cell.In one embodiment, the electrogenic activity does not require theaddition of a redox mediator as compared to a conventional microbialfuel cell. In one embodiment, the alcohologenic biocatalyst is not an“opportunistic” pathogen as that term is understood in the art.

In one embodiment, the processes described above are scalable up 10, 100to 1000 times or more for large-scale ethanol and electricityproduction. In one embodiment, the electricity generated can be used toreplace some of the electricity demand of a biofuel production facility,such as an ethanol and/or biodiesel production facility. In oneembodiment, electricity is produced using a bioelectrochemical cell(BEC). In one embodiment, the ethanol produced according to the methodsdescribed herein can be distilled in a biodiesel production facilityusing existing distillation equipment and reused as the alcohol in thetransesterification reaction.

Embodiments described herein include computer-implemented systems andmethods operating according to particular functions or algorithms whichmay be implemented in software or a combination of software and humanimplemented procedures. In one embodiment, the software may comprisecomputer executable instructions stored on computer readable media suchas memory or other type of storage devices. Further, such functionscorrespond to modules, which are software, hardware, firmware or anycombination thereof. Multiple functions may be performed in one or moremodules as desired, and the embodiments described are merely examples.The software may be executed on a digital signal processor, ASIC,microprocessor, or other type of processor operating on a computer,i.e., a computer system, such as a personal computer, server or othercomputer system.

Embodiments of the invention will be further described by reference tothe following examples, which are offered to further illustrate variousembodiments of the present invention. It should be understood, however,that many variations and modifications may be made while remainingwithin the scope of the present invention.

Example 1

Preliminary experiments were performed to select a CBP organism and toshow that byproducts of ethanol fermentation produced by a CBP organismfrom lignocellulosic substrates can be converted into CO₂ and electronsby an electricigen. In these experiments, the lignocellulosic substratewas AFEX-treated corn stover (hereinafter termed AFEX-CS), the CBPorganism was C. uda and the electricigen used was G. sulfurreducens(Gsu) which catalyzed the conversion of fermentation byproducts using achemical electron acceptor (fumarate). These experiments alsodemonstrated that rates of ethanol production were increased by at leastten (10)% up to about 15% during co-culture growth through the removalof fermentation byproducts whose accumulation would otherwise inhibitthe consolidated bioprocessing step and/or ethanologenesis.

Equipment

Liquid fermentation byproducts such as ethanol and organic acids insupernatant fluids were analyzed by in a High Performance LiquidChromatography (HPLC) system equipped with a 25P pump running at 0.6mL/min and 1600 PSI, in-line degasser AF, 2487 dual wavelengthabsorbance detector, 410 Differential refractometer, and 717Plusautosampler (Waters, Milford, Mass.) and a standard Cartridge Holder#125-0131 with a 30×4.6 mm Micro-Guard Carbo-C Refill Cartridgeconnected to an Aminex HPX-87H Ion exclusion column(Bio-Rad). The columnwas heated at temperature 25° C. Approximately 100 μl of sample wasinjected for analyses and metabolite separation was achieved using in acarrier solution of 4 mM H₂SO₄ in ddH₂O. Data acquisition was with aMicrosoft Compaq computer equipped with Breeze software (Waters,Milford, Mass.).Gaseous fermentation byproducts such as H₂ and CO₂ wereanalyzed in a CP-4900 Micro Gas Chromatograph (Varian, Inc, Palo Alto,Calif.) equipped with a MSAH BF column with >99.999% Argon carrier gasand a PPQ column with >99.99% Helium carrier gas. Data collection waswith a Dell Latitude D620 computer running Galaxie Chromatography DataSystem version 1.9.3.2 (Varian, Inc, Palo Alto, Calif.).

Starting Materials

Chemicals

All chemicals were from Sigma-Aldrich and had a minimum purity of 98%.For Gsu growth, sodium acetate and sodium fumarate were routinely usedas electron donor and acceptor, respectively. CBP organisms wereroutinely grown with sugars such as cellobiose, glucose and xylose.

Substrate

Ammonia Fiber Expansion (“AFEX”) treated corn stover (everythingremaining after grain is harvested, typically including stalks andleaves w/o cobs) was used as the substrate in this testing. AFEX-CS wasprovided from Dr. Dale's Laboratory, Michigan State University (EastLansing, Mich.). It was prepared from corn stover (CS), premilled andpassed through a 4 mm screen, provided by the National Renewable EnergyLaboratory (NREL, Golden, Colo.). The moisture content of the untreatedCS was about 7% (total weight basis). Feedstock analysis by NRELrevealed an estimated composition (dry weight basis) of 34.1% cellulose,22.8% xylan, 4.2% arabinan, 11.4% lignin and 2.3% protein in the cornstover. The milled CS was kept at 4° C. for long-term storage. The AFEXpretreatment was conducted in a 2.0 L pressure vessel (Parr) equippedwith thermocouples and a pressure sensor. The vessel was heated to 100to 110° C. before 240 g of prewetted CS at 60% moisture (dry weightbasis) was loaded. The lid was bolted shut. Concurrently, 150 ganhydrous ammonia was added to a separate 500 ml stainless steelcylinder (Parker Instrumentation) and heated until the gas pressurereached 4.48 MPa (650 psi). Heated ammonia was then transferred into thereactor to initiate the reaction. After 15 min, the pressure wasreleased through an exhaust valve. The initial and final temperatures ofthe pretreatment were 130±5° C. and 110±5° C., respectively. After AFEXtreatment, pretreated CS was air-dried overnight under a fume hood, andkept at 4° C. for long-term storage. Approximately 125 g of AFEX-CS weresupplied to this laboratory in a one-gallon ZIPLOCK bag and stored atfour (4) ° C. The AFEX-CS was ground in a grinder (GE Model 168940) andsieved through a ceramic filter with 0.75 mm×0.75 mm pores.

Consolidated Bioprocessing (CPB) Organisms

Clostridium cellulolyticum, Clostridium hungatei AD, Clostridiumhungatei B3B, Clostridium papyrosolvens C7, Ruminococcus albus, C. udaATCC 21399, Cellulomonas biazotea, Cellulomonas cartae, Cellulomonasgelida, Cellulomonas fimi, Cellulomonas uda ATCC 491, Cellulomonasflavigena, Cellulomonas cellobioparum, Clostridium longisporum,Clostridium populeti, Clostridium cellulovorans, Clostridiumphytofermentans, Clostridium lentocellum, C. papyrosolvens NCIMB fromthe inventors' laboratory culture collection were used.

These strains originated from a laboratory culture collection at theUniversity of Massachusetts (Amherst, Mass.). The strains were grown inGS2 medium, as described in “Cellulase system of a free-living,mesophilic clostridium (strain C7)” K Cavedon, S B Leschine and ECanale-Parola J Bacteriol. 1990 August; 172(8): 4222-4230, which isincorporated herein by reference in its entirety, with 0.2% cellobioseor 0.2% glucose as the sole carbon and energy source. The strains wereincubated at 30° C. Frozen stocks in 10% dimethyl sulfoxide weremaintained at −80° C. for long-term storage.

Acetivibrio cellulolyticus ATCC 33288 (Ace) was purchased from TheAmerican Type Culture Collection ATCC (Manassas, Va.) for use in thesepreliminary experiments. It was cultured in a medium known as ATCC 1207(ATCC, Manassas, Va.) and incubated at 37° C.

Electricigen

G. sulfurreducens ATCC 51573 (Gsu) was used as the electricigen. Thismicrobe was obtained originally from the American Type CultureCollection (ATCC) as a substantially pure culture and maintained underconditions known in the art in the inventors' laboratory culturecollection.

Procedure

The various organisms were screened for ethanologenic efficiency in“Regan's medium,” see Regan, supra. AFEX-CS (0.2%) was added as sourceof carbon and energy. Fermentation efficiencies were assessed by firstquantifying the ethanol yields and then the yields of liquid (organicacids) and gaseous (hydrogen and CO₂) fermentation byproducts in one totwo week cultures via HPLC or GC analyses.

For co-culture experiments between Cuda and Gsu, strains were pre-grownto mid to late exponential phase in Regan's medium with, respectively,0.2% D+ cellobiose or 15 mM acetate and 40 mM fumarate and incubated at30° C. in a rotating drum incubator (Glas-Col 099A RD4512) run at lowspeed (10 percent). Approximately a 10% (v/v) inoculum with an opticaldensity at 660 nm of 0.2 was added to anaerobic (N₂:CO₂. 80:20) pressuretubes (Bellco) containing 10 ml of Regan's medium and 0.2% AFEX-CS.Controls with Cuda alone (with or without 40 mM fumarate), Gsu alone,and uninoculated controls also were included. All cultures tubes wereincubated at 30° C. in a rotating drum incubator, as described above.

Growth was monitored periodically by taking the optical density of thecultures at 660 nm. For growth measurements, the tubes were removed fromthe rotating incubator and the AFEX-CS was allowed to settle to thebottom of the tube for ˜10 minutes prior to measuring the opticaldensity of the culture. The uninoculated control tubes were used tocalibrate the absorbance readings to zero.

The headspace of each tube was sampled periodically (every 1-3 days) toanalyze the gas composition (hydrogen and CO₂) by gas chromatography(GC). Approximately 1 mL of headspace was removed with a N₂-flushedsyringe and injected into the GC. Supernatant samples (0.7 ml) were alsoremoved anaerobically, 70 μl were used to plate on solid Regan's mediumsupplemented with 0.2% cellobiose and measure growth of Cuda as colonyforming units. The rest of the sample was filtered through a 0.45 μMmembrane filter (Fisher) and stored at −20° C. before being analyzed byHPLC.

Initial Screening and Results

After one-to-two weeks of incubation at 30° C. the ethanologenicefficiency of each organism was assessed by measuring the yields ofethanol in cell-free supernatant fluids. The best ethanologens (ethanolyields more than 40% of the maximum theoretical yields) were C.populeti, C. lentocellum, A. cellulolyticus, C. gelida, C. biazotea, andC. uda ATCC 21399. The other strains were discarded.

Further Screening and Results

The liquid (organic acids) and gaseous (hydrogen gas and CO2)fermentation byproducts from the selected ethanologenic strains weremeasured by HPLC and GC analyses, respectively. Based on the predominantfermentation byproduct (hydrogen or organic acids) the strains in thistesting were two functional categories.

The first group (C. populeti, C. lentocellum, and A. cellulolyticus)produced H₂ as their main fermentation product.

C. populeti was discarded because growth studies using soluble sugarssuch as 0.2% glucose revealed abrupt cell lysis as the culture reachedcell densities more than 0.6 units of absorbance at 600 nm, which isconsistent with the presence of lytic phage infection in this organism.Lytic phage infections have been reported in the genus Clostridium andare a major source of contamination in industrial fermentations.Infected strains are difficult to cure of the virus and are notdesirable for industrial processes.

Otherwise, as FIG. 3 shows, Ace produced significantly more H₂ than C.lentocellum during the degradation of AFEX-CS and during thefermentation of the cellulose repeating disaccharide unit, cellobiose.Acetate and formate were the predominant organic acids produced duringfermentation of AFEX-CS by both Ace and Clen (not shown).

The second group (C. gelida, C. biazotea, and C. uda ATCC 21399)consisted of members of the family Cellulomonadaceae, a group offacultative aerobic actinobacteria, and produced predominantly organicacids (mainly acetate and/or formate) as fermentation products, withvery little or undetectable fermentative hydrogen gas.

The high level of organic acid production by the organisms in this groupled to media acidification during fermentation, thereby causingsuboptimal growth and fermentation. In the case of C. uda, the pH of themedium dropped from 7 to 5.5 during the degradation and fermentation ofAFEX-CS, Thus, removal of organic acids is expected to have a positiveeffect in cell growth and ethanologenesis.

As compared with Cuda, C. lentocellum had a higher growth rate duringthe co-fermentation of glucose and xylose, as shown in FIG. 4.

Specifically, the growth rates for the fermentation of single sugars wassix- and three-fold higher for C. lentocellum as compared with Cuda whengrown, with 0.2% glucose and 0.2% xylose, respectively, which makes C.lentocellum a more robust strain for industrial fermentations of theindividual sugars.

However, the co-fermentation rates of Cuda and Clen were comparable, asshown in FIG. 4, which makes them attractive candidates forfermentations based on lignocellulose substrates.

In addition, Cuda was the only cellulomonad that grew optimally underthe anaerobic conditions useful for optimum fuel cell performance. Cudaalso produced the highest yields of acetate and formate, namely morethan 20 mM and 30 mM, respectively. For this reason, it was selected forfurther studies.

Testing Fermentation Byproducts Removal by Gsu

Based on the above results, removal of organic acids to support growthof Gsu was predicted to prevent media acidification in cultures of Cudagrown with AFEX-CS and therefore increase cell viability and ethanolyields. To test this hypothesis, Cuda with Gsu were co-cultivated inculture vessels with AFEX-CS as sole source of carbon and energy, withfumarate as an electron acceptor for growth of Gsu. Negative controlswith Gsu alone (which could not grow with AFEX-CS as an electron donorand fumarate as an electron acceptor) and in co-culture with Cuda but inmedia without fumarate (which serves as electron acceptor for growth ofGsu) were used to demonstrate that any metabolic changes were aconsequence of consortium synthrophic growth.

As FIG. 5 shows, when grown in co-culture, the rates of biomassdegradation by Cuda increased approximately 10 to 15%, as did theethanol yields of Gsu. The growth of Cuda also was stimulated more than15-fold as fermentation byproducts were removed by Gsu, as measured bythe increase in Cuda's colony forming units. The growth of both Cuda andGsu also is shown as absorbance at 660 nm in FIG. 5. In addition, allfermentation byproducts (organic acids and hydrogen) were used by Gsu,in a process coupled to the reduction of fumarate in the medium, whichwas monitored by the accumulation of succinate. CO₂ produced from thecomplete oxidation of acetate was used as a proxy for Gsu growth, whichincreased during co-cultivation of the two strains.

Conclusion

These results demonstrate that coupling an appropriate CBP organism toan electricigen works effectively to remove fermentation byproducts andincrease the rates of biomass degradation and bioethanol production.These results further demonstrated that removal of organic acidseffectively increased growth of Cuda. However, the amount of ethanol didnot increase linearly.

Of all the organisms tested, C. populeti, C. lentocellum, A.cellulolyticus, C. gelida, C. biazotea, and C. uda ATCC 21399 were amongthe best ethanologens.

Cuda, along with the clostridial strain C. lentocellum described above,had the highest ethanol yields from AFEX-CS.

Cuda and C. lentocellum also had the highest growth rates during theanaerobic co-fermentation of six- and five-carbon sugars.

Co-culturing of Cuda with Gsu stimulated the growth of the CBP organismwith AFEX-CS and the yields of ethanol while supporting the removal offermentation byproducts and electron transfer by Gsu.

Example 2 Optimization of a MFC

A Precision Mechanical Convection incubator (cat no. 51220098) wascustomized to house electronic equipment and electrical cords connectinga customized MFC described below to an external potentiostat (Bio-LogicUSA,VSP model) and connected to a Dell Inspiron 1721 laptop computerrunning the EC-Lab software V9.55 (Bio-Logic USA), which controlled theelectrochemical parameters of the MFC and stored the output data.

An H-type, two-chambered microbial fuel cell was built and tested asshown in FIG. 2 and described above. The chamber volumes (100 ml) werereduced by 5-times the volume of standard H-type fuel cells (asdescribed in Improved fuel cell and electrode designs for producingelectricity from microbial degradation, Park D H, Zeikus J G. BiotechnolBioeng. 2003 Feb. 5; 81(3):348-55), which is incorporated herein byreference in its entirety, to minimize the costs associated with eachrun, maintain anaerobiosis and improve reproducibility. The fuel cellswere also designed such that both the anode and cathode of four (4) fuelcells could be stirred with a single 10-place stir plate (FisherScientific, IKAMAG) enabling four experiments to be carried outsimultaneously.

Several anode and cathode electrode configurations were tested. Aplatinum wire can be used as the cathode electrode, however cheapergraphite materials were tested in order to minimize costs and increaseelectrode surface area. The electrode materials tested were graphitewith different degrees of porosity (i.e., fine woven graphite felt(Electrosynthesis), porous graphite blocks, and graphite cylinders (>99%purity, Alfa Aesar), which were tested to find the most inexpensivematerial that could produce reproducible results and be reusable. Thegraphite cylinders had the lowest resistance of the electrode materials(i.e. less than 0.5Ω) and were the most durable so they were used forthe anode and cathode electrodes.

Three types of connectors were tested for use with the graphitecylinders: 0.5 mm platinum wire (Electrosynthesis), 0.5 mm copper wire,and commercially available water-tight glass-reinforced epoxy connectors(Teledyne Impulse, XSA-BC) which connect to an approximately two (2) ftstranded wire (Teledyne Impulse, RMA-FS). Conductive silver epoxy(Fulton Radio, Inc.) was used to seal the connections. The commerciallyavailable connectors performed the best because they were inexpensive,created a tight seal to protect the wire and silver epoxy connectionsfrom corrosion, were the most durable, and allowed the electrodes to beremoved from the fuel cell for further analysis (e.g. confocalmicroscopy). The completed electrodes had resistances of less than 0.5Ω.

Three different types of growth media were tested in the optimized fuelcell configuration, namely fresh water (FW), Regan media and Daniel Bondmedia (“DB”). All growth media were supplemented with 1-15 mM acetate.No current was produced when the Gsu culture was initiated in Reganmedia. Current was produced from FW and DB, however the conversionefficiency in DB was greater than 70% while the conversion for FW was˜20%. Additionally, DB media supported the growth of Gsu as well as manyof the tested CBP organisms, including Cuda, and so was chosen as thepreferred fuel cell media.

Gsu cell inoculation conditions were also optimized. The cells weregrown in FW+ Ferric citrate media supplemented with 15 mM acetate (SeeDevelopment of a Genetic System for Geobacter sulfurreducens. Coppi M V,Leang C, Sandler S J, and Lovley D R. Applied and EnvironmentalMicrobiology. 2001 July; Vol. 67(7): 3180-3187, which is incorporatedherein by reference in its entirety), FW media supplemented with 15 mMacetate and 40 mM fumarate and DB media supplemented with 15-20 mMacetate and 40 mM fumarate.

The 40% vol/vol of cells (i.e. 36 mL) were harvested at late exponentialphase, centrifuged to remove the excess acetate and fumarate, washed andthen resuspended in fuel cell media and inoculated into the anodechamber. The cells from the DB media started producing current afterapproximately 18 hours while the cells from FW took approximately 24hours, and the cells from FW+ ferric citrate took approximately 5 hoursbut did not proceed into exponential growth phase until approximately 48hours. DB media was therefore chosen as the inoculation media for theGsu cells because they performed well and because it is convenient touse the same media for all stages of the fuel cell setup.

Inoculation conditions were further optimized by testing the currentproduction rates from cells grown to exponential phase and those grownto early stationary phase. Cells inoculated from stationary phase startproducing current sooner (at approximately 12 hrs) so this inoculationcondition was chosen.

Details of the optimized double chamber MFC are in Example 3 below.

Example 3

Unless otherwise indicated, all materials (including AFEX-CS) used wereas described in Examples 1 and 2 above.

Microbial Fuel Cell (MFC)

The MFC optimized as described above in Example 2, was constructed andused in this testing. For convenience, reference numbers are directed tothe exemplary MFC 118 shown in FIG. 2, although FIG. 2 is not to beinterpreted as limited to the specific sizes, materials andconfigurations of the components described in this example.

The anode and cathode chambers (e.g., 204 and 205), respectively, wereconstructed from 100-mL Pyrex media bottles (Fisher Scientific).Custom-made glass side ports were fused to the bottles (Michigan StateUniversity glass shop) using 18-mm diameter glass pressure tubes(Bellco). The various ports (e.g., 220A, 220B, 222A and 222B) weresealed with 18-mm septum stoppers (Fisher Scientific).

The electrodes (e.g., 206 and 207) used in the MFC were 2.5 cm-longgraphite rods, each having removable water-sealed connectors, to allowfor removal of the wires from the electrode when needed, for example, toexamine the microbial biofilm formed on the electrode by microscopy orto clean them after each use.

A glass bridge (comprising, for example, the cation exchange membrane210, gaskets 211 and glass flanges 212) was constructed with a 15-mmdiameter glass tube and a 32-mm diameter glass flange. A 32-mm diameterNAFION cation exchange membrane (Ion Power, Inc. N117) was sandwichedbetween two 32-mm diameter rubber gaskets (purchased at a local hardwarestore and modified to have a 15-mm hole in the middle). The glassflanges allowed for passage of H+ ions, but also served to keep thecontents of each chamber separated. The NAFION membrane was cut intosmall circles, sandwiched between the rubber gaskets and sealed withepoxy between the two chambers. The NAFION membrane, rubber gaskets andglass flanges assembly were held together with a metal joint pinch clamp(Thomas Scientific).

The anode and cathode electrodes were constructed from 2.5 cm×1.3 cmgraphite rods (Alfa Aesar), which were drilled to house aglass-reinforced epoxy connector (Teledyne Impulse, XSA-BC). Silverepoxy (Fulton Radio, Inc.) was used to make a tight conductive sealbetween the graphite and the connector such that the electrodes had lessthan 0.5Ω resistance. The imbedded connector was connected via awater-tight seal to a rubber-molded connector with an approximately two(2) ft stranded wire (Teledyne Impulse, RMA-FS).

The anode and cathode wires were imbedded into a No. 6 rubber stopper atthe mouth of each chamber. Between experiments, the graphite electrodeswere refreshed by soaking briefly in 1N HCl to remove trace metals, and1N NaOH to remove organic material. The graphite was then polished with400 grit sandpaper and rinsed in double distilled H₂O.

An Ag/AgCl reference electrode (Bioanalytical systems, Inc. MF-2078) wasplaced in the anode chamber using the sparging port (e.g., 222A). Thisenabled the potential of the anode electrode (e.g., 206) to becontrolled using the potentiostat (Bio-Logic USA,VSP model). The cableof the reference electrode (e.g., 213C) was embedded through a hole inthe septum stopper of the sparging port. The hole was sealed withwaterproof silicone (General Electric).

Consolidated Bioprocessing (“CBP”) Organism

The CBP organism Cuda was selected for these experiments based on itsgrowth robustness with AFEX-CS under anaerobic conditions, ability toco-ferment six- and five-carbon sugars, ethanologenic yields (>40%maximum theoretical yield), and range of fermentation byproducts(acetate, formate, and to a lesser extent, H₂) that can serve aselectron donors for the electricigenic bacterium (in the case of Gsu),as described in Example 1.

For these MFC experiments, C. uda was previously grown for 24 h in DBmedia with 0.2% cellobiose at 30° C. 36 mL (40% vol/vol) of cells wereharvested by centrifugation, resuspended in DB medium containing theAFEX-CS, and inoculated into the anode chamber 204.

Electricigen

G. sulfurreducens ATCC 51573 (Gsu) was used as the electricigen. In thisform, the Gsu is known to efficiently convert fermentation products(such as H₂, acetate, and formate) to electricity in MFCs andelectrochemical cells. As the testing in Example 1 and below describes,growth rates of Gsu were unaffected in the presence of AFEX-CS.Furthermore, as shown in Example 1, this organism was capable of growingin co-culture with Cuda and coupling the conversion of all thefermentation byproducts to the reduction of a chemical electron acceptorsuch as fumarate. Thus, it was hypothesized that it could also couplethe transfer of electrons from fermentation byproducts to the anodeelectrode of a MFC driven by AFEX-CS.

Testing

Conditions for growing Gsu in the MFC with acetate (1-6 mM) as electrondonor were optimized as described in Example 2 and are described indetail under Procedures. These are conditions that enabled reproducibleand relatively “fast” electricity production (i.e., startingapproximately one (1) to two (2) hrs after inoculation of the MFC 118with the electricigen Gsu) and also had the highest coulombicefficiencies (70 to 75% of acetate converted into electricity). Electrondonor-to-current conversion efficiencies (coulombic efficiencies) arecalculated using the EC-lab software from the integral of the currentproduction curve over time to obtain the total coulombs transferred tothe anode electrode during electricity production. The moles of electrondonor (i.e., acetate) used by the electricigen are calculated bymeasuring its concentration in culture supernatant fluid samples byHPLC, as described in Example 1. The following equation is then used tocalculate coulombic efficiency:

$C_{E} = \frac{\overset{t}{\int\limits_{0}}{Idt}}{{Fbv}_{an}\Delta \; c}$

I=current in coulombs per secondt=time of experimentF=Faraday's constantb=the mole of electrons exchanged per mole of substratev_(an)=volume of the anode compartmentΔc=concentration of substrate in mol/L

Controls with AFEX-CS and Gsu produced no current while controls withAFEX-CS/Gsu/acetate (3 mM) produced current with yields and coulombicefficiencies comparable to the Gsu/acetate (3 mM) (not shown).

Cuda controls growing alone with AFEX-CS also were included.

Consortia-Driven Electrochemical Cells

As shown in FIG. 6, when grown with three (3) mM acetate in a MFC Gsuproduced current from acetate. When substantially all of the acetate wasused, the current declined sharply. Once the current ceased, AFEX-CS andCuda were added to the anode chamber and the current resumedimmediately, reaching outputs approximately twice those obtained withthe one (1) mM acetate. Ethanol and fermentation byproducts as well asany sugars remaining from the degradation of corn stover were measureddaily.

Growth of the co-culture in the fuel cell was tested with and withoutnitrogen gas sparging. The use of nitrogen gas sparging facilitated thediffusion of organic acids through the anode biofilms and increased theyields of electricity. It also helped remove volatile solvents such asethanol from the medium via gas stripping, thus removing possiblesolvent tolerance issues potentially capable of compromising theviability of Cuda and Gsu at the maximum theoretical production ofethanol desired for scaled-up applications.

As predicted, co-culturing converted much of the organic acids intoelectricity, but did not increase ethanol production. Fermentation wasalso inefficient, as some of the sugars were not used when compared toCuda controls (grown alone with AFEX-CS) or co-cultures in whichsparging was maintained throughout the experiment. Sparging of themedium with N₂ gas removed the ethanol as it was being produced, leadingto a nearly 100% fermentation efficiency, an approximately 1.6-foldincrease in electricity production, and removal of substantially all theformate and most of the acetate (at least about 15 mM) which wereconverted into electricity. The theoretical prediction estimates anapproximately two-fold increase in ethanol production under theseconditions (or the equivalent of more than 80% of the maximumtheoretical yield).

Cuda control cultures grown alone with AFEX-CS had a 100% fermentationefficiency with no detectable levels of soluble sugars in the medium andsubstantially all the theoretical electron content of the glucose andxylose components of the AFEX-corn stover (13.91 mmol of electrons)accounted for as acetate (21%), formate (37%), and ethanol (43%) (SeeFIG. 7).

Fermentation byproducts are known to negatively affect the rates ofhydrolysis and fermentation by CBP organisms. Specifically, hydrogen isknown to be a feedback inhibitor of cellulose hydrolysis, while organicacids quickly lead to media acidification, thereby decreasing growthrobustness and fermentation yields.

Removal of the organic acids helped to support the growth of Gsu andcurrent production and also prevented media acidification. In controlcultures with Cuda and AFEX-CS alone, the pH dropped to 5.5 but remainedclose to neutral in the MFCs with the co-cultures or with Gsu alone. Asa result of pH stability, cell viability and ethanol yields are expectedto increase.

Strain Improvement for Increased Performance

Use of genetic engineering approaches for manipulation of the nature ofthe metabolic capabilities of the consortium partners were alsoinvestigated. Such approaches could potentially be used to customize thebioprocessing scheme and control the biofuel and electricity ratiosproduced using this platform. To accomplish this, a mutant of Gsucarrying a deletion in the genes encoding the uptake-hydrogenase Hyb ofGsu (hereinafter termed Gsu Hyb) was obtained from Dr. Coppi'sLaboratory (University of Massachusetts, Amherst) and tested inco-culture with Cuda using AFEX-CS as substrate for Cuda and fumarate asterminal electron acceptor for Gsu Hyb. Controls with co-cultures ofCuda and genetically unaltered Gsu were used for comparison.

Hydrogen, even when present at very low levels or concentration (i.e.,less than 1 mM) was determined to be preferentially used as an electrondonor by Gsu anode biofilms during electricity production. A molecule ofH₂ provides 2 electrons for power generation while acetate provides 8.Thus, we hypothesized that growth of Gsu and indirectly electricitygeneration could be increased when co-culturing a mutant of Gsu unableto use H₂ as an electron donor but able otherwise to use organic acids.

As bars 804 show in FIG. 8, although Cuda (“A”) produces low levels ofH₂ during fermentation, an approximately 1.2-fold increase in the growthof the Gsu Hyb mutant strain (“B) in consortium with Cuda was observed.Ethanol yields (802) remained substantially the same, however. Theseresults demonstrate that it is possible to genetically engineer Gsu tomanipulate the nature of the metabolic interaction, e.g., interspeciesorganic acid transfer, including with interspecies H₂-transfer, betweenthe consortia partners to increase the overall energetic output of thesystem.

Taken together, these results demonstrate that fuel cells powered by anelectricigen and a CBP organism can be effectively used as platforms forcellulosic ethanol production.

Procedure

The potential of the anode (working) electrode was controlled by thepotentiostat, which was connected via wires (e.g., 213A, 213B and 213C)and alligator clips to the anode electrode, the cathode electrode andthe reference electrode (e.g., 206, 207 and 216, respectively). Thepotentiostat was connected to a PC computer equipped with EC-labsoftware (Biol-Logic USA), which allowed real-time monitoring of thecurrent produced at the anode electrode 202. The potentiostat 216 hasfour potentiostatic/galvanostatic boards so that four electrochemicalcells 118 can be run simultaneously.

While the experiment was running, the anode and cathode chambers weresparged continuously with anaerobic gases by connecting gas distributionlines (Norprene tubing, Cole Parmer) to a Luer Lok hose end adapter(Fisher Scientific) and then to a 23-gauge needle 214 (BenctonDickinson). Sterile 0.22-um syringe filters (Fisher Scientific) wasplaced between the Luer Lok hose adapter and the needle to sterilize thegases. Needles (e.g., 223A and 223B) were inserted through the septumstoppers of the sparging ports (e.g., 222A and 222B).

Gas outlets, e.g., 220A and 220B, were added to the anode and cathodechambers to release the CO₂ (produced during the conversion of organicacids into electricity by the electricigen) and the H₂ (produced fromthe electrochemical reaction of electrons and proton at the cathode),respectively, as well as any gas (N₂ and CO₂) used to sparge the growthmedium (e.g., 208). The gas outlets were constructed using 12-cm metalcannulas (Popper), which were placed through stoppers (e.g., 218A and218B) that sealed the top openings of the anode and cathode chambers.The top of the gas outlets (i.e., the cannulas) were each attached to aone-way female to male stopcock (Fisher Scientific) to open or close therespective gas outlets as needed.

Stirring also was achieved with ½″×⅛″ octagonal stirbars (FisherScientific), which were located in the anode and cathode chambers, byplacing the chambers on a 10-place stirplate (Fisher Scientific,IKAMAG).

The electrochemical cell experiments were run in the incubator(Described in Example 1) so that the temperature could be controlled tosupport the growth of the microbial consortia. The Cuda/Gsu consortiumused in this testing was incubated at 30° C.

The electrochemical cell, set up as described above, but without thereference electrode or media, was autoclaved to sterilize it. Thereference electrode was then sterilized by immersion in 70% ethanol,allowed to dry and added aseptically to the anode chamber. 90 mL ofanaerobic DB media (prepared as described in Example 2) was addedaseptically to the anode 204 and cathode 208 chambers (herein called DBmedia). Acetate (1 mM) was added to the anode 204 chamber to initiatethe growth of the electricigen 120. The electrochemical cells areincubated at 30° C. and sparged with anaerobic gas mix (N₂:CO₂, 80:20)to buffer the pH of the medium at 7. Stirring was initiated at 500 rpm.

An electricigen 120 was grown as a film on the anode electrode 208located in the anode chamber 202. The anode electrode 202 was poised atthe desired potential (an anode potential of +0.24V was used to make acathode-unlimited system) with respect to the Ag/AgCl referenceelectrode 214 using a potentiostat 212. In this way, a cathode-unlimitedsystem was used for controlled and reproducible results. The system wasallowed to equilibrate for a minimum of approximately two hours.

The electricigen 120 (Gsu) was subcultured at approximately 30° C. in100-ml of standard anaerobic DB media supplemented with the desiredconcentration of electron donor (e.g. 10-30 mM acetate) and electronacceptor (e.g., 40-50 mM fumarate). Cells from 36 ml (or 40% of thevolume of the anode chamber 204) of early stationary phase cultures(e.g., those that have ceased exponential growth) of the electricigen120 Gsu were harvested by centrifugation (6,000 rpm, 8 min, fixed rotor,25° C.), washed once with DB media without acetate or fumarate, andresuspended in DB media. The cells were then injected aseptically intothe anode chamber 204 with a syringe and a needle and through theside-arm septum port 222. Samples of the media in the anode chamber 204were periodically removed with a syringe and a needle and through theside-arm septum port 222 for HPLC analyses of organic acids and sugars.

Current production was initiated after approximately 12-18 hours hadpassed from the initiation of the experiment by adding approximately one(1) mM of acetate as the electron donor. The current increasedexponentially in the next 24-48 hr until substantially all the acetatewas used by the electricigen 120. Current generation was determined tobe directly related to the growth of the electricigen as a film on theanode electrode 202. In the examples presented here, substantially allthe acetate was consumed in 40-48 h after measurable current wasinitiated and an orange electricigenic film 120 was visually apparent onthe anode electrode 202. Once the acetate was used, the current declinedsharply.

The CBP organism and the AFEX-CS were added to the anode chamber oncethe current reached zero (0) mA.

Example 4

The equipment and starting materials as described in the above exampleswere used herein. FIGS. 9-13 T show how conversion efficiencies andcurrent yields are affected depending on the type of inoculationprocedure followed.

Sequential Inoculation

In one experiment, a CBP organism (Cuda) was added to a film of anelectricigen (Gsu) (which was pre-grown on the anode electrode with 1 mMacetate) at the top of current production, during current decline onceall of the acetate has been used, or when current ceased (0 mA), asshown in FIG. 9. Sparging was used to facilitate mixing while theelectricigen was forming a film on the anode. Once the CBP organism wasadded, the sparging and outlet ports of the anode chamber were closed.

In the experiments shown in FIG. 9 ethanol yields in the range of 40-50%and yields of conversion of fermentation byproducts into electricity inthe range of 8-13% of the maximum theoretical yields were reached,respectively, after 24, 26, and 22 h. of inoculation with the insolublesand the CBP organism. This contrasts with the more than 80% ethanolyields reached when sparging was maintained throughout the experiment,as shown in FIG. 8, to evaporate the ethanol produced by the CBPorganism and substantially minimize growth inhibition of the CBPorganism due to the accumulation of ethanol.

These results show that biocatalysts, such as C. uda and the AFEX-CS,can be added sequentially at any given time during current production bythe electricigen. In this example, current recovery was shown when theAFEX-CS and C. uda were added at various times, when Gsu is at itsmaximum current (‘at top’, dashed line), when it is half way in decline(‘at decline’, double line) and when current has reached 0 mA (‘at 0current’, thick black line). (See FIG. 9).

These results also show that improving the ethanol tolerance of the CBPorganism is expected to substantially increase ethanol yields at levelsmore than 80% of the maximum theoretical.

Substantially Simultaneous Inoculation

In this experiment, biocatalysts (Cuda and Gsu) were added substantiallysimultaneously, together with the insolubles (AFEX-CS) into the anodechamber. One (1) mM of acetate was added to one of the inoculums to jumpstart the growth of Gsu's growth. Sparging was maintained throughout theexperiment to evaporate the ethanol and promote growth of theelectricigen on the anode electrode.

As FIG. 10 shows current generation starts after 2-4 hours of incubationand increases exponentially after 9-14 h in cultures with or withoutacetate supplementation, respectively. Maximum currents are reachedafter 40-46 h in both experiments.

FIG. 11 shows the ethanologenic efficiency of the organisms tested inFIG. 10, which are of at least about 80% of the maximum theoreticalyield, with all the fermentation byproducts (acetate, formate, lactateand H₂) having been converted into electricity. This is in contrast tothe ethanologenic efficiency of controls of Cuda alone, also shown inFIG. 11, which produced less than 40% of the maximum theoretical yield.

FIG. 12 is a bar graph showing the conversion efficiency of fermentationbyproducts into electricity for the sequential and simultaneousinoculations of FIGS. 9 and 10, respectively, in embodiments of thepresent invention.

FIG. 13 is a bar graph showing maximum current yields for the sequentialand simultaneous inoculations of FIGS. 9 and 10, respectively, inembodiments of the present invention

These results show that simultaneous addition of the biocatalysts doesnot affect conversion efficiencies. Additionally, supplementation withacetate allowed Gsu to generate current more quickly and increasedcurrent yields. Adding components simultaneously also minimizesdisruptions to the bioprocessing reaction and reduces costs associatedwith the set up of the bioreactor.

Conclusions

As FIGS. 9-13 show, simultaneous inoculation had a higher efficiency atconverting fermentation byproducts into electricity as compared tosequential inoculation, but was a slower process (reduced rates perday). Addition of acetate is slightly less efficient but faster. Thus,inoculation strategies can be used to customize the yields and rates ofelectricity generation.

Inoculation at mid-point of current decline appears to be optimal forboth parameters when using sequential inoculation.

Example 5 Identification of a Glycerol-Fermenting Microbial Catalyst.

Gram-positive bacteria, Clostridia, (from culture collection identifiedin Example 1) was selected for use in ethanologenic fermentation ofglycerol. Cultures were supplemented with 0.6% yeast extract to mimicthe reported sugar content of biodiesel wastewater, which alsocontributes to the fermentative metabolism, ethanologenesis, andgeneration of fermentation byproducts.

Among the more than 10 species tested, only C. cellobioparum (Cce) grewwell, consumed substantially all the glycerol (0.25% (w/v) from solutionand fermented it to ethanol, acetate, lactate and formate and H₂. SeeFIGS. 15A and 15B. Ethanol was the major fermentation product (ca. 40%of maximum theoretical yield) and was not produced in the same mediumwithout glycerol, thus confirming it was produced from glycerolfermentation. Cce's growth rates and yields were naturally robust,suggesting that this organism is naturally tuned for fermentative growthand ethanologenesis from glycerol. It also produced only fermentationbyproducts that can serve as electron donors for the electricigen G.sulfurreducens (Gsu). For this reason, Cce was selected as thefermentative catalyst for the microbial platform.

Stimulation of Glycerol Fermentation in the Co-Culture

Removal of H₂ and organic acids during coculture of Cce with Gsu greatlystimulated fermentative growth as shown in FIG. 15A. The acetate,formate and H₂ were removed by Gsu by providing fumarate as an electronacceptor. However, lactate remained in the fermentation broth. As aresult, lactate accumulation dropped the pH of the fermentation broth to6.3, which also negatively affected the growth of Gsu and Cce.

Strain Improvement by Adaptive Evolution

Glycerol tolerance of the microbial catalysts was also tested, with theresults shown in FIGS. 16A-16C. The fermentative catalyst, Cce, grewwith up to 7% (w/v) glycerol while maintaining growth rates of at least75% of the maxima (FIG. 16A). After successive passages with increasingconcentrations of glycerol, an alcohol-tolerant strain of Cce (CceA)adapted for growth with 10% glycerol (FIG. 16B) was produced. The growthof the electrogenic partner, Gsu, was inhibited at 7% glycerol in boththe monoculture and the coculture (FIG. 16A).

Robustness of Gsu was improved by selecting for variants that grew withinhibitory (1%) concentrations of ethanol (FIG. 16C). After six monthsof successive passages at increasing ethanol concentrations, analcohol-tolerant strain of Gsu (GsuA) was isolated, which tolerated 4%ethanol (FIG. 16C). GsuA also increased its glycerol tolerance to 10%glycerol in both the monoculture and CceA coculture (FIG. 16B).

Despite the improved glycerol tolerance in CceA, glycerol consumptionreached a plateau once ethanol production reached levels ca. 0.5%.Hence, the alcohol tolerance of CceA was investigated. Strainsensitivity to ethanol concentrations in this range was confirmed (FIG.16C).

Coupling Glycerol Fermentation to Current Production in a BEC.

The coupling of glycerol fermentation and ethanologenesis to currentproduction was demonstrated in a BEC driven by the CceA-GsuA co-culture.For these experiments, GsuA was incubated at 30° C. in the anode chamberof an anoxic, dual-chamber, H-type MFC equipped with graphite rodelectrodes poised at a constant potential of 240 mV. This anoxic, poisedsystem maintained consistency between different fuel cells, removed anypotential limitations resulting from electron transfer at the cathode,and eliminated the possibility of oxygen intrusion into the anodechamber that might support aerobic growth.

When 1 mM acetate was added to the medium in the anode chamber, thecurrent rapidly increased to ca. 1 mA and then declined as the acetatewas depleted (FIG. 17). The coulombic efficiency of GsuA was comparableto Gsu (89.8±3.3%), demonstrating that evolving alcohol tolerance hadnot affected its electrogenic activity.

Once current production declined, CceA was added to the GsuA anodechamber at location 1702 and current production resumed. Glycerolconsumption and ethanol production were stimulated (2.8- and 1.4-fold,respectively) in the BECs driven by the co-culture of CceA-GsuA, ascompared to CceA controls.

As observed previously, ethanol production reached a plateau once itreached growth inhibitory concentrations (0.6%). Although current(3.2±0.3 mmol of electrons) was produced from fermentation byproducts,it declined before all of the acetate and formate was removed. BecauseGsuA grows well at these alcohol concentrations, it is unlikely that theinefficient removal of electron donors was caused by ethanol inhibition(FIG. 16C). However, lactate also accumulated in the BEC broth andacidified the medium (final pH of 5.5). In fact, the electrogenicefficiency of GsuA declined once the pH began to drop below 6. Theseresults demonstrate that BECs can be used to stimulate glycerolfermentation while generating ethanol and current.

Conclusion

The composition of the final, 80% concentrated crude glycerin varies.See, for example, Suehara, K. et al. Biological treatment of wastewaterdischarged from biodiesel fuel production plant with alkali-catalyzedtransesterification. J. Biosci. Bioeng. 100, 437-442,doi:S1389-1723(05)70489-8 [pii] 10.1263/jbb.100.437 (2005) and Williams,P. R., Inman, D., Aden, A. & Heath, G. A. Environmental andsustainability factors associated with next-generation biofuels in theU.S.: what do we really know? Environ. Sci. Technol. 43, 4763-4775(2009), both of which are incorporated herein by reference. Based onthese reports, calculations understood by those skilled in the art weremade to determine the content of glycerol and alcohol in glycerinwastewater (after acid pretreatment to remove oil and precipitate thesoap and salts). These calculations showed that glycerin wastewaterlikely contains about 10 to about 20% glycerol and about 2 to about 6%alcohol. These values are within the ranges reported forlaboratory-treated raw glycerol solutions derived from several biodieselplants. See, for example, Moon, C., Ahn, J. H., Kim, S. W., Sang, B. I.& Um, Y. Effect of biodiesel-derived raw glycerol on 1,3-propanediolproduction by different microorganisms. Appl. Biochem. Biotechnol. 161,502-510, doi:10.1007/s12010-009-8859-6 (2010), which is incorporatedherein by reference. As such, these values provide confirmation that theminimum alcohol target has been reached for GsuA and the glycerol targetfor both strains.

Adaptively evolving the catalysts to improve their alcohol tolerance isexpected to further improve performance. As shown in this example, analcohol-tolerant strain of Gsu, termed GsuA, was evolved and grew in thepresence of 4% (v/v) ethanol and tolerated 10% glycerol loadings. It isexpected that catalyst performance can be even further enhanced viaadaptive evolution and/or genetic engineering to further increasealcohol and glycerol tolerances. Such approaches are also expected toco-evolve glycerol productivity and ethanol yields.

Example 6

Cultures from Example 5 were transferred in the stationary phase, whenerror-prone DNA Polymerase IV is expressed and the mutation rate ishigh. See, for example, Tompkins, J. D. et al. Error-prone polymerase,DNA polymerase IV, is responsible for transient hypermutation duringadaptive mutation in Escherichia coli. J. Bacteriol. 185, 3469-3472(2003), which is incorporated herein by reference. Transfer during thisphase increases the potential for mutant variants to arise andaccelerates an otherwise slow process.

Gsu was transferred in this manner at higher concentrations of ethanoland variants growing with 4.5% ethanol have been demonstrated. Thus,this approach is expected to be effective to reach a targeted industrialconcentration of 6% in glycerin wastewater or higher.

Example 7

CceA was transferred in this manner at higher concentrations of ethanoland variants growing with 2% ethanol have been demonstrated.

These results suggest that the approach being used is rapid andeffective. Inasmuch as ethanol sensitivity limited glycerol fermentationby CceA, alcohol tolerance is also expected to increase fermentationrobustness and ethanol yields from 10% (w/v) glycerol.

Example 8 (Prophetic)

The hypothesis noted in Example 7 will be monitored by periodicallygrowing the alcohol-tolerant variants in cultures with 10% glycerol andmeasuring glycerol consumption and ethanol production in thefermentation broth.

Example 9 (Prophetic)

Growth of the transferred cultures (such as Gsu in Example 6 and CceA inExample 7) will be monitored as optical density of the cultures at 660nm using a spectrophotometer. Once growth is observed at a targetalcohol concentration, the cultures will be maintained through severalpassages in the same ethanol concentration until growth rates and yieldsreturn to native, unchallenged levels or until they stabilize.

At this point, aliquots of cultures containing the adapted variants willbe plated to isolate individual colonies, corresponding to clonalvariants. Approximately 10 colonies will be inoculated in fresh liquidmedium with ethanol to identify the fastest-growing variants. Afterrepeated passages in exponential phase, the fastest growers are enrichedand will be preserved anaerobically at −80° C. in dimethyl sulfoxide(DMSO). The variant with the best growth rates and yields will betransferred to the next concentration increment (0.5%) to initiate a newround of evolution. The experiment will end when variants no longerarise.

It is expected that concentrations of ethanol at or above the 6% targetwill be achieved.

Example 10 (Prophetic)

Additional testing will include testing of industrial solid loadings(above 2%), of other CBP organisms and electricigen combinations, strainimprovement through genetic engineering and adaptive evolution, testingof other substrates, and producing biofuels other than ethanol.

Conclusion

The embodiments described herein provide an economically andenvironmentally superior consolidated bioprocessing technology forethanol and electrical power production from substrates in abioelectrochemical cell, such as a microbial fuel cell, as compared toconventional microbially-catalyzed consolidated bioprocessing. Based oncurrent estimates, CBP bioprocessing is expected to reduce the cost ofcellulosic ethanol by as much as 51%, and provide ethanol yields closeto 90% (or more) of the maximum theoretical yields, while theco-fermentation of both glucose and xylose is expected to reduce thefinal fuel cost by at least an additional six (6)%. CBP processing isexpected to reduce the cost of biodiesel and provide similar ethanolyields, while the co-fermentation of both glucose and xylose is expectedto reduce the final cost by at least an additional six (6)%.

In one embodiment, a maximum yield of ethanol above 80% and asubstantially complete co-fermentation of six- and five-carbon sugarswhile producing electricity as a value-added product is produced. In oneembodiment, feedstock processing and diversification strategies areintegrated in a single step fermentation, with removal of non-valued,inhibitory products for power co-generation. In one embodiment, strainimprovement by genetic engineering and direct evolution is utilized.

In one embodiment, a single-chambered electrochemical bioreactor systemcan be used for biofuel production, which can be constructed usingcommercially available bioreactors and fermentors.

Unlike conventional methods, embodiments described herein decouplebioenergy production from the food supply and reduces processing coststhrough the use of low cost lignocelluloses substrates, single-stephydrolysis and fermentation, and conversion of low-value fermentationbyproducts into electricity. In one embodiment, the conversions takeplace in a single bioreactor or vessel, thereby minimizing costsassociated with chemical separations of fermentation products anddevelopment of secondary processing units.

The embodiments described herein, which directly generate electricityand/or ethanol from glycerin-containing water, such as a glycerin streamfrom biodiesel product, provide an inexpensive alternative to glycerinwastewater refining. The process not only provides for treatment of theglycerin wastewater, but also generates biofuels and energy, which canbe sold, transported or used in the biodiesel production facility.

Embodiments described herein provide a competitive consolidatedbioprocessing technology for biofuel and electrical power production ina microbial fuel cell or electrochemical cell. The novel processesdescribed herein integrate feedstock processing and diversificationstrategies, single step hydrolysis and fermentation, use of fermentationbyproducts for electricity generation, and microorganism strainimprovement through genetic engineering and direct evolution.

In one embodiment, the novel system and methods described herein arecustomized for other types of biomass and/or other types of biofuel, byselecting a particular CBP organism and electricigenic partner. In oneembodiment, genetic engineering and adaptive evolution of thesebacterial partners is used to modulate biofuel and electricityproduction rates and yields.

Although specific embodiments have been illustrated and describedherein, it will be appreciated by those of ordinary skill in the artthat any procedure that is calculated to achieve the same purpose may besubstituted for the specific embodiments shown. This application isintended to cover any adaptations or variations of the present subjectmatter. For example, although the fermentation byproducts have beendescribed as including primarily organic acids and/or primarilyhydrogen, it is to be understood that other fermentation byproducts mayalso be useful herein. Therefore, it is manifestly intended thatembodiments of this invention be limited only by the claims and theequivalents thereof.

1-21. (canceled)
 22. A method comprising: a consolidated hydrolyzing andfermentation step for converting a biomass to a biofuel with a firstorganism in an anode chamber, wherein the anode reactor contains ananode electrode and the converting step produces a fermentationbyproduct; transferring electrons in the byproduct to the anodeelectrode with a second organism to produce a film; and allowing thefilm to catalytically split the electrons and protons, wherein theelectrons flow towards a cathode electrode to produce electricity andthe protons permeate a proton-exchange membrane connecting the anodechamber and the cathode chamber, wherein the electrons and protons reactto produce hydrogen gas.
 23. The method of claim 22 wherein theelectricigen is G. sulfurreducens (Gsu).
 24. The method of claim 23where the first and second organisms are added sequentially.
 25. Themethod of claim 23 wherein the first and second organisms are addedsubstantially simultaneously.
 26. The method of claim 22 furthercomprising applying a potential to the anode electrode.
 27. The methodof claim 23 wherein the first organism comprises one or morecellulomonads.
 28. The method of claim 22 wherein the biofuel isethanol.
 29. The method of claim 22 wherein the biofuel is biodieselfuel.
 30. The method of claim 22 wherein the biomass is a non-foodbiomass.
 31. The method of claim 30 wherein the non-food biomass is cornstover.
 32. The method of claim 31 wherein the non-food biomass isglycerin-containing water.
 33. A method comprising: a consolidatedhydrolyzing and fermentation step for converting a biomass to a productwith one or more mesophilic consolidated bioprocessing organism in areaction chamber containing the biomass, a second organism comprising anelectricigen disposed on an anode electrode contained in the reactionchamber, wherein the converting step produces one or more byproducts;transferring electrons in said byproducts to the anode electrode throughthe second organism comprising an electricigen; and allowing the secondorganism to catalytically split electrons and protons from saidbyproducts, wherein the electrons flow from the anode to a cathodeelectrode located in the reaction chamber to produce electricity and theelectrons and the protons react to produce hydrogen gas.
 34. The methodof claim 33 wherein the cathode chamber and the anode chamber comprise asingle chamber, wherein the single chamber further comprises a referenceelectrode.
 35. The method of claim 34 wherein the anode electrode, thecathode electrode and the reference electrode are electronicallyconnected to each other and to an external electric current whichcreates a potential between the anode and cathode.
 36. The method ofclaim 33 wherein the anode chamber and cathode chamber are separated bya proton exchange membrane, wherein the anode chamber further comprisesa reference electrode.
 37. The method of claim 33 wherein the secondorganism is disposed on the anode electrode as a film.
 38. The method ofclaim 33 wherein the electrons and the protons react at the cathodeelectrode.
 39. The method of claim 33 wherein the product is a biofuel.40. The method of claim 39 wherein the biofuel is ethanol or biodieselfuel.
 41. The method of claim 33 wherein the one or more byproductscomprises one or more fermentation byproducts.